<?xml version="1.0" encoding="us-ascii"?>
<!-- DFIN ActiveDisclosure(SM) XBRL Linkbase Document - http://www.dfinsolutions.com/ -->
<!-- Creation Date      : 2020-11-12T21:28:09.6906074+00:00 -->
<!-- Version            : 5.0.1.513 -->
<!-- Package ID         : a86503685f4941519e70b36abfa05fb9 -->
<!-- Copyright (c) 2020 Donnelley Financial Solutions, Inc. All Rights Reserved. -->
<link:linkbase xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd">
  <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all" arcroleURI="http://xbrl.org/int/dim/arcrole/all" />
  <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default" arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" />
  <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain" arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" />
  <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member" arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" />
  <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension" arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" />
  <link:roleRef xlink:type="simple" xlink:href="astc-20200930.xsd#TemplateLink" roleURI="http://www.astrotechcorp.com/20200930/role/TemplateLink" />
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/StatementCondensedConsolidatedBalanceSheets" xlink:href="astc-20200930.xsd#Role_StatementCondensedConsolidatedBalanceSheets" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/StatementCondensedConsolidatedBalanceSheets">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LiabilitiesAndStockholdersEquity" xlink:type="locator" xlink:label="us-gaap_LiabilitiesAndStockholdersEquity" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockholdersEquity" xlink:type="locator" xlink:label="us-gaap_StockholdersEquity" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RetainedEarningsAccumulatedDeficit" xlink:type="locator" xlink:label="us-gaap_RetainedEarningsAccumulatedDeficit" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AdditionalPaidInCapital" xlink:type="locator" xlink:label="us-gaap_AdditionalPaidInCapital" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_TreasuryStockValue" xlink:type="locator" xlink:label="us-gaap_TreasuryStockValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockValue" xlink:type="locator" xlink:label="us-gaap_CommonStockValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PreferredStockValue" xlink:type="locator" xlink:label="us-gaap_PreferredStockValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockholdersEquityAbstract" xlink:type="locator" xlink:label="us-gaap_StockholdersEquityAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommitmentsAndContingencies" xlink:type="locator" xlink:label="us-gaap_CommitmentsAndContingencies" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_Liabilities" xlink:type="locator" xlink:label="us-gaap_Liabilities" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_OtherLiabilitiesNoncurrent" xlink:type="locator" xlink:label="us-gaap_OtherLiabilitiesNoncurrent" />
    <link:loc xlink:href="astc-20200930.xsd#astc_OperatingAndFinanceLeaseLiabilityNoncurrent" xlink:type="locator" xlink:label="astc_OperatingAndFinanceLeaseLiabilityNoncurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LongTermNotesPayable" xlink:type="locator" xlink:label="us-gaap_LongTermNotesPayable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LiabilitiesCurrent" xlink:type="locator" xlink:label="us-gaap_LiabilitiesCurrent" />
    <link:loc xlink:href="astc-20200930.xsd#astc_OperatingAndFinanceLeaseLiabilityCurrent" xlink:type="locator" xlink:label="astc_OperatingAndFinanceLeaseLiabilityCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NotesPayableCurrent" xlink:type="locator" xlink:label="us-gaap_NotesPayableCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NotesPayableRelatedPartiesClassifiedCurrent" xlink:type="locator" xlink:label="us-gaap_NotesPayableRelatedPartiesClassifiedCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AccruedIncomeTaxesCurrent" xlink:type="locator" xlink:label="us-gaap_AccruedIncomeTaxesCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AccruedLiabilitiesAndOtherLiabilities" xlink:type="locator" xlink:label="us-gaap_AccruedLiabilitiesAndOtherLiabilities" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EmployeeRelatedLiabilitiesCurrent" xlink:type="locator" xlink:label="us-gaap_EmployeeRelatedLiabilitiesCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AccountsPayableCurrent" xlink:type="locator" xlink:label="us-gaap_AccountsPayableCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LiabilitiesCurrentAbstract" xlink:type="locator" xlink:label="us-gaap_LiabilitiesCurrentAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:type="locator" xlink:label="us-gaap_LiabilitiesAndStockholdersEquityAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_Assets" xlink:type="locator" xlink:label="us-gaap_Assets" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_OtherAssetsNoncurrent" xlink:type="locator" xlink:label="us-gaap_OtherAssetsNoncurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_OperatingLeaseRightOfUseAsset" xlink:type="locator" xlink:label="us-gaap_OperatingLeaseRightOfUseAsset" />
    <link:loc xlink:href="astc-20200930.xsd#astc_AssetsHeldForSaleNonCurrent" xlink:type="locator" xlink:label="astc_AssetsHeldForSaleNonCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PropertyPlantAndEquipmentNet" xlink:type="locator" xlink:label="us-gaap_PropertyPlantAndEquipmentNet" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AssetsCurrent" xlink:type="locator" xlink:label="us-gaap_AssetsCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PrepaidExpenseAndOtherAssetsCurrent" xlink:type="locator" xlink:label="us-gaap_PrepaidExpenseAndOtherAssetsCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncomeTaxesReceivable" xlink:type="locator" xlink:label="us-gaap_IncomeTaxesReceivable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_InventoryFinishedGoods" xlink:type="locator" xlink:label="us-gaap_InventoryFinishedGoods" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_InventoryWorkInProcess" xlink:type="locator" xlink:label="us-gaap_InventoryWorkInProcess" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_InventoryRawMaterialsNetOfReserves" xlink:type="locator" xlink:label="us-gaap_InventoryRawMaterialsNetOfReserves" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_InventoryNetAbstract" xlink:type="locator" xlink:label="us-gaap_InventoryNetAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AccountsReceivableNetCurrent" xlink:type="locator" xlink:label="us-gaap_AccountsReceivableNetCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:type="locator" xlink:label="us-gaap_CashAndCashEquivalentsAtCarryingValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AssetsCurrentAbstract" xlink:type="locator" xlink:label="us-gaap_AssetsCurrentAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AssetsAbstract" xlink:type="locator" xlink:label="us-gaap_AssetsAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ConvertiblePreferredStockMember" xlink:type="locator" xlink:label="us-gaap_ConvertiblePreferredStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfStockDomain" xlink:type="locator" xlink:label="us-gaap_ClassOfStockDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfStockDomain" xlink:type="locator" xlink:label="us-gaap_ClassOfStockDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementClassOfStockAxis" xlink:type="locator" xlink:label="us-gaap_StatementClassOfStockAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementTable" xlink:type="locator" xlink:label="us-gaap_StatementTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementLineItems" xlink:type="locator" xlink:label="us-gaap_StatementLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" order="10320.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" order="10350.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain_2" order="10360.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" order="10380.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_ConvertiblePreferredStockMember" order="10400.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AssetsAbstract" order="10430.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="us-gaap_AssetsCurrentAbstract" order="10510.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_CashAndCashEquivalentsAtCarryingValue" order="10590.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_AccountsReceivableNetCurrent" order="10630.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_InventoryNetAbstract" order="10670.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_InventoryNetAbstract" xlink:to="us-gaap_InventoryRawMaterialsNetOfReserves" order="10750.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_InventoryNetAbstract" xlink:to="us-gaap_InventoryWorkInProcess" order="10790.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_InventoryNetAbstract" xlink:to="us-gaap_InventoryFinishedGoods" order="10830.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_IncomeTaxesReceivable" order="10870.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_PrepaidExpenseAndOtherAssetsCurrent" order="10910.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_AssetsCurrent" order="10950.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="us-gaap_PropertyPlantAndEquipmentNet" order="10990.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="astc_AssetsHeldForSaleNonCurrent" order="11030.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="us-gaap_OperatingLeaseRightOfUseAsset" order="11070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="us-gaap_OtherAssetsNoncurrent" order="11110.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="us-gaap_Assets" order="11150.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LiabilitiesAndStockholdersEquityAbstract" order="11190.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:to="us-gaap_LiabilitiesCurrentAbstract" order="11270.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_AccountsPayableCurrent" order="11350.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_EmployeeRelatedLiabilitiesCurrent" order="11390.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_AccruedLiabilitiesAndOtherLiabilities" order="11430.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_AccruedIncomeTaxesCurrent" order="11470.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_NotesPayableRelatedPartiesClassifiedCurrent" order="11510.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_NotesPayableCurrent" order="11550.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="astc_OperatingAndFinanceLeaseLiabilityCurrent" order="11590.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_LiabilitiesCurrent" order="11630.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:to="us-gaap_LongTermNotesPayable" order="11670.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:to="astc_OperatingAndFinanceLeaseLiabilityNoncurrent" order="11710.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:to="us-gaap_OtherLiabilitiesNoncurrent" order="11750.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:to="us-gaap_Liabilities" order="11790.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:to="us-gaap_CommitmentsAndContingencies" order="11830.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:to="us-gaap_StockholdersEquityAbstract" order="11870.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_PreferredStockValue" order="11950.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_CommonStockValue" order="11990.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_TreasuryStockValue" order="12030.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_AdditionalPaidInCapital" order="12070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_RetainedEarningsAccumulatedDeficit" order="12110.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_StockholdersEquity" order="12150.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:to="us-gaap_LiabilitiesAndStockholdersEquity" order="12190.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/StatementCondensedConsolidatedBalanceSheetsParenthetical" xlink:href="astc-20200930.xsd#Role_StatementCondensedConsolidatedBalanceSheetsParenthetical" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/StatementCondensedConsolidatedBalanceSheetsParenthetical">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_TreasuryStockShares" xlink:type="locator" xlink:label="us-gaap_TreasuryStockShares" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockSharesOutstanding" xlink:type="locator" xlink:label="us-gaap_CommonStockSharesOutstanding" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockSharesIssued" xlink:type="locator" xlink:label="us-gaap_CommonStockSharesIssued" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockSharesAuthorized" xlink:type="locator" xlink:label="us-gaap_CommonStockSharesAuthorized" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockNoParValue" xlink:type="locator" xlink:label="us-gaap_CommonStockNoParValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PreferredStockSharesOutstanding" xlink:type="locator" xlink:label="us-gaap_PreferredStockSharesOutstanding" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PreferredStockSharesIssued" xlink:type="locator" xlink:label="us-gaap_PreferredStockSharesIssued" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PreferredStockSharesAuthorized" xlink:type="locator" xlink:label="us-gaap_PreferredStockSharesAuthorized" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PreferredStockNoParValue" xlink:type="locator" xlink:label="us-gaap_PreferredStockNoParValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SeriesDPreferredStockMember" xlink:type="locator" xlink:label="us-gaap_SeriesDPreferredStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ConvertiblePreferredStockMember" xlink:type="locator" xlink:label="us-gaap_ConvertiblePreferredStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfStockDomain" xlink:type="locator" xlink:label="us-gaap_ClassOfStockDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementClassOfStockAxis" xlink:type="locator" xlink:label="us-gaap_StatementClassOfStockAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementTable" xlink:type="locator" xlink:label="us-gaap_StatementTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementLineItems" xlink:type="locator" xlink:label="us-gaap_StatementLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_ConvertiblePreferredStockMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_SeriesDPreferredStockMember" order="10130.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockNoParValue" order="10180.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockSharesAuthorized" order="10220.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockSharesIssued" order="10260.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_PreferredStockSharesOutstanding" order="10300.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockNoParValue" order="10340.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesAuthorized" order="10380.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesIssued" order="10420.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesOutstanding" order="10460.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TreasuryStockShares" order="10500.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/StatementCondensedConsolidatedStatementOfChangesInStockholdersEquityDeficit" xlink:href="astc-20200930.xsd#Role_StatementCondensedConsolidatedStatementOfChangesInStockholdersEquityDeficit" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/StatementCondensedConsolidatedStatementOfChangesInStockholdersEquityDeficit">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ProfitLoss" xlink:type="locator" xlink:label="us-gaap_ProfitLoss" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardGross" xlink:type="locator" xlink:label="us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardGross" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardGross" xlink:type="locator" xlink:label="us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardGross" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardForfeited" xlink:type="locator" xlink:label="us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardForfeited" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardForfeitures" xlink:type="locator" xlink:label="us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardForfeitures" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationAndExerciseOfStockOptions" xlink:type="locator" xlink:label="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationAndExerciseOfStockOptions" />
    <link:loc xlink:href="astc-20200930.xsd#astc_StockOfferingCosts" xlink:type="locator" xlink:label="astc_StockOfferingCosts" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="locator" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:type="locator" xlink:label="us-gaap_StockIssuedDuringPeriodValueNewIssues" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AccountingStandardsUpdateExtensibleList" xlink:type="locator" xlink:label="us-gaap_AccountingStandardsUpdateExtensibleList" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SharesOutstanding" xlink:type="locator" xlink:label="us-gaap_SharesOutstanding" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest" xlink:type="locator" xlink:label="us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:type="locator" xlink:label="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_CumulativeEffectPeriodOfAdoptionAdjustmentMember" xlink:type="locator" xlink:label="srt_CumulativeEffectPeriodOfAdoptionAdjustmentMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_CumulativeEffectPeriodOfAdoptionDomain" xlink:type="locator" xlink:label="srt_CumulativeEffectPeriodOfAdoptionDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_CumulativeEffectPeriodOfAdoptionDomain" xlink:type="locator" xlink:label="srt_CumulativeEffectPeriodOfAdoptionDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_CumulativeEffectPeriodOfAdoptionAxis" xlink:type="locator" xlink:label="srt_CumulativeEffectPeriodOfAdoptionAxis" />
    <link:loc xlink:href="astc-20200930.xsd#astc_SeriesDConvertiblePreferredStockMember" xlink:type="locator" xlink:label="astc_SeriesDConvertiblePreferredStockMember" />
    <link:loc xlink:href="astc-20200930.xsd#astc_SeriesCConvertiblePreferredStockMember" xlink:type="locator" xlink:label="astc_SeriesCConvertiblePreferredStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfStockDomain" xlink:type="locator" xlink:label="us-gaap_ClassOfStockDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementClassOfStockAxis" xlink:type="locator" xlink:label="us-gaap_StatementClassOfStockAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RetainedEarningsMember" xlink:type="locator" xlink:label="us-gaap_RetainedEarningsMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AdditionalPaidInCapitalMember" xlink:type="locator" xlink:label="us-gaap_AdditionalPaidInCapitalMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_TreasuryStockMember" xlink:type="locator" xlink:label="us-gaap_TreasuryStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockMember" xlink:type="locator" xlink:label="us-gaap_CommonStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EquityComponentDomain" xlink:type="locator" xlink:label="us-gaap_EquityComponentDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EquityComponentDomain" xlink:type="locator" xlink:label="us-gaap_EquityComponentDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementEquityComponentsAxis" xlink:type="locator" xlink:label="us-gaap_StatementEquityComponentsAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementTable" xlink:type="locator" xlink:label="us-gaap_StatementTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementLineItems" xlink:type="locator" xlink:label="us-gaap_StatementLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementEquityComponentsAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain_2" order="10050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_TreasuryStockMember" order="10130.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AdditionalPaidInCapitalMember" order="10150.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_RetainedEarningsMember" order="10170.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" order="10180.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" order="10210.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="astc_SeriesCConvertiblePreferredStockMember" order="10230.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="astc_SeriesDConvertiblePreferredStockMember" order="10270.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_CumulativeEffectPeriodOfAdoptionAxis" order="10300.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_CumulativeEffectPeriodOfAdoptionAxis" xlink:to="srt_CumulativeEffectPeriodOfAdoptionDomain_2" order="10310.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_CumulativeEffectPeriodOfAdoptionAxis" xlink:to="srt_CumulativeEffectPeriodOfAdoptionDomain" order="10330.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_CumulativeEffectPeriodOfAdoptionDomain" xlink:to="srt_CumulativeEffectPeriodOfAdoptionAdjustmentMember" order="10350.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" order="10700.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockholdersEquityIncludingPortionAttributableToNoncontrollingInterest" order="11140.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_SharesOutstanding" order="11160.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_AccountingStandardsUpdateExtensibleList" order="11580.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodValueNewIssues" order="11800.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" order="12020.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="astc_StockOfferingCosts" order="12240.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_AdjustmentsToAdditionalPaidInCapitalSharebasedCompensationAndExerciseOfStockOptions" order="12460.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardForfeitures" order="12680.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardForfeited" order="12780.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodValueRestrictedStockAwardGross" order="12900.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_StockIssuedDuringPeriodSharesRestrictedStockAwardGross" order="13000.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInStockholdersEquityRollForward" xlink:to="us-gaap_ProfitLoss" order="13120.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/StatementCondensedConsolidatedStatementsOfCashFlows" xlink:href="astc-20200930.xsd#Role_StatementCondensedConsolidatedStatementsOfCashFlows" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/StatementCondensedConsolidatedStatementsOfCashFlows">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RightOfUseAssetObtainedInExchangeForOperatingLeaseLiability" xlink:type="locator" xlink:label="us-gaap_RightOfUseAssetObtainedInExchangeForOperatingLeaseLiability" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AccountingStandardsUpdateExtensibleList" xlink:type="locator" xlink:label="us-gaap_AccountingStandardsUpdateExtensibleList" />
    <link:loc xlink:href="astc-20200930.xsd#astc_AdjustmentToRetainedEarningsFromAdoptionOfAccountingStandardUpdate" xlink:type="locator" xlink:label="astc_AdjustmentToRetainedEarningsFromAdoptionOfAccountingStandardUpdate" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncomeTaxesPaid" xlink:type="locator" xlink:label="us-gaap_IncomeTaxesPaid" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_InterestPaidNet" xlink:type="locator" xlink:label="us-gaap_InterestPaidNet" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SupplementalCashFlowInformationAbstract" xlink:type="locator" xlink:label="us-gaap_SupplementalCashFlowInformationAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:type="locator" xlink:label="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" xlink:type="locator" xlink:label="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NetCashProvidedByUsedInFinancingActivities" xlink:type="locator" xlink:label="us-gaap_NetCashProvidedByUsedInFinancingActivities" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:type="locator" xlink:label="us-gaap_ProceedsFromIssuanceOfCommonStock" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ProceedsFromRelatedPartyDebt" xlink:type="locator" xlink:label="us-gaap_ProceedsFromRelatedPartyDebt" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:type="locator" xlink:label="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NetCashProvidedByUsedInInvestingActivities" xlink:type="locator" xlink:label="us-gaap_NetCashProvidedByUsedInInvestingActivities" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PaymentsToAcquirePropertyPlantAndEquipment" xlink:type="locator" xlink:label="us-gaap_PaymentsToAcquirePropertyPlantAndEquipment" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:type="locator" xlink:label="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NetCashProvidedByUsedInOperatingActivities" xlink:type="locator" xlink:label="us-gaap_NetCashProvidedByUsedInOperatingActivities" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncreaseDecreaseInOtherOperatingCapitalNet" xlink:type="locator" xlink:label="us-gaap_IncreaseDecreaseInOtherOperatingCapitalNet" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncreaseDecreaseInAccountsPayable" xlink:type="locator" xlink:label="us-gaap_IncreaseDecreaseInAccountsPayable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncreaseDecreaseInInventories" xlink:type="locator" xlink:label="us-gaap_IncreaseDecreaseInInventories" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncreaseDecreaseInIncomeTaxesReceivable" xlink:type="locator" xlink:label="us-gaap_IncreaseDecreaseInIncomeTaxesReceivable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncreaseDecreaseInAccountsReceivable" xlink:type="locator" xlink:label="us-gaap_IncreaseDecreaseInAccountsReceivable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:type="locator" xlink:label="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_GainLossOnDispositionOfAssets1" xlink:type="locator" xlink:label="us-gaap_GainLossOnDispositionOfAssets1" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DepreciationDepletionAndAmortization" xlink:type="locator" xlink:label="us-gaap_DepreciationDepletionAndAmortization" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensation" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensation" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="locator" xlink:label="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ProfitLoss" xlink:type="locator" xlink:label="us-gaap_ProfitLoss" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="locator" xlink:label="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_CumulativeEffectPeriodOfAdoptionAdjustmentMember" xlink:type="locator" xlink:label="srt_CumulativeEffectPeriodOfAdoptionAdjustmentMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_CumulativeEffectPeriodOfAdoptionDomain" xlink:type="locator" xlink:label="srt_CumulativeEffectPeriodOfAdoptionDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_CumulativeEffectPeriodOfAdoptionAxis" xlink:type="locator" xlink:label="srt_CumulativeEffectPeriodOfAdoptionAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementTable" xlink:type="locator" xlink:label="us-gaap_StatementTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementLineItems" xlink:type="locator" xlink:label="us-gaap_StatementLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" order="10160.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="srt_CumulativeEffectPeriodOfAdoptionAxis" order="10190.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_CumulativeEffectPeriodOfAdoptionAxis" xlink:to="srt_CumulativeEffectPeriodOfAdoptionDomain" order="10220.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_CumulativeEffectPeriodOfAdoptionDomain" xlink:to="srt_CumulativeEffectPeriodOfAdoptionAdjustmentMember" order="10240.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" order="10290.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_ProfitLoss" order="10370.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" order="10410.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_ShareBasedCompensation" order="10490.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_DepreciationDepletionAndAmortization" order="10530.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_GainLossOnDispositionOfAssets1" order="10570.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" order="10610.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInAccountsReceivable" order="10690.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInIncomeTaxesReceivable" order="10730.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInInventories" order="10770.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInAccountsPayable" order="10810.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInOtherOperatingCapitalNet" order="10850.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInOperatingActivities" order="10890.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" order="10930.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="us-gaap_PaymentsToAcquirePropertyPlantAndEquipment" order="11010.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInInvestingActivities" order="11050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" order="11090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_ProceedsFromRelatedPartyDebt" order="11170.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_ProceedsFromIssuanceOfCommonStock" order="11210.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInFinancingActivities" order="11250.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" order="11290.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" order="11330.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SupplementalCashFlowInformationAbstract" order="11410.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SupplementalCashFlowInformationAbstract" xlink:to="us-gaap_InterestPaidNet" order="11490.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SupplementalCashFlowInformationAbstract" xlink:to="us-gaap_IncomeTaxesPaid" order="11530.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SupplementalCashFlowInformationAbstract" xlink:to="astc_AdjustmentToRetainedEarningsFromAdoptionOfAccountingStandardUpdate" order="11570.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SupplementalCashFlowInformationAbstract" xlink:to="us-gaap_AccountingStandardsUpdateExtensibleList" order="11610.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SupplementalCashFlowInformationAbstract" xlink:to="us-gaap_RightOfUseAssetObtainedInExchangeForOperatingLeaseLiability" order="11650.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureGeneralInformationAdditionalInformationDetails" xlink:href="astc-20200930.xsd#Role_DisclosureGeneralInformationAdditionalInformationDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureGeneralInformationAdditionalInformationDetails">
    <link:loc xlink:href="astc-20200930.xsd#astc_AdditionalFutureServiceAndSupportCommitments" xlink:type="locator" xlink:label="astc_AdditionalFutureServiceAndSupportCommitments" />
    <link:loc xlink:href="astc-20200930.xsd#astc_PurchaseOrders" xlink:type="locator" xlink:label="astc_PurchaseOrders" />
    <link:loc xlink:href="astc-20200930.xsd#astc_NumberOfWhollyOwnedSubsidiaries" xlink:type="locator" xlink:label="astc_NumberOfWhollyOwnedSubsidiaries" />
    <link:loc xlink:href="astc-20200930.xsd#astc_NumberOfAdditionalPatentsInProcess" xlink:type="locator" xlink:label="astc_NumberOfAdditionalPatentsInProcess" />
    <link:loc xlink:href="astc-20200930.xsd#astc_NumberOfPatentsGranted" xlink:type="locator" xlink:label="astc_NumberOfPatentsGranted" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsequentEventMember" xlink:type="locator" xlink:label="us-gaap_SubsequentEventMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsequentEventTypeDomain" xlink:type="locator" xlink:label="us-gaap_SubsequentEventTypeDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsequentEventTypeDomain" xlink:type="locator" xlink:label="us-gaap_SubsequentEventTypeDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsequentEventTypeAxis" xlink:type="locator" xlink:label="us-gaap_SubsequentEventTypeAxis" />
    <link:loc xlink:href="astc-20200930.xsd#astc_GeneralInformationTable" xlink:type="locator" xlink:label="astc_GeneralInformationTable" />
    <link:loc xlink:href="astc-20200930.xsd#astc_GeneralInformationLineItems" xlink:type="locator" xlink:label="astc_GeneralInformationLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="astc_GeneralInformationLineItems" xlink:to="astc_GeneralInformationTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="astc_GeneralInformationTable" xlink:to="us-gaap_SubsequentEventTypeAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain_2" order="10050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventTypeDomain" xlink:to="us-gaap_SubsequentEventMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_GeneralInformationLineItems" xlink:to="astc_NumberOfPatentsGranted" order="10140.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_GeneralInformationLineItems" xlink:to="astc_NumberOfAdditionalPatentsInProcess" order="10160.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_GeneralInformationLineItems" xlink:to="astc_NumberOfWhollyOwnedSubsidiaries" order="10180.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_GeneralInformationLineItems" xlink:to="astc_PurchaseOrders" order="10230.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_GeneralInformationLineItems" xlink:to="astc_AdditionalFutureServiceAndSupportCommitments" order="10250.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureGoingConcernAdditionalInformationDetails" xlink:href="astc-20200930.xsd#Role_DisclosureGoingConcernAdditionalInformationDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureGoingConcernAdditionalInformationDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ProceedsFromIssuanceOrSaleOfEquity" xlink:type="locator" xlink:label="us-gaap_ProceedsFromIssuanceOrSaleOfEquity" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsequentEventMember" xlink:type="locator" xlink:label="us-gaap_SubsequentEventMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsequentEventTypeDomain" xlink:type="locator" xlink:label="us-gaap_SubsequentEventTypeDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsequentEventTypeAxis" xlink:type="locator" xlink:label="us-gaap_SubsequentEventTypeAxis" />
    <link:loc xlink:href="astc-20200930.xsd#astc_RegisteredOfferingPurchaseAgreementMember" xlink:type="locator" xlink:label="astc_RegisteredOfferingPurchaseAgreementMember" />
    <link:loc xlink:href="astc-20200930.xsd#astc_PublicOfferingPurchaseAgreementMember" xlink:type="locator" xlink:label="astc_PublicOfferingPurchaseAgreementMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="locator" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="locator" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_TypeOfArrangementAxis" xlink:type="locator" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockMember" xlink:type="locator" xlink:label="us-gaap_CommonStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EquityComponentDomain" xlink:type="locator" xlink:label="us-gaap_EquityComponentDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementEquityComponentsAxis" xlink:type="locator" xlink:label="us-gaap_StatementEquityComponentsAxis" />
    <link:loc xlink:href="astc-20200930.xsd#astc_OrganizationConsolidationAndPresentationOfFinancialStatementsTable" xlink:type="locator" xlink:label="astc_OrganizationConsolidationAndPresentationOfFinancialStatementsTable" />
    <link:loc xlink:href="astc-20200930.xsd#astc_OrganizationConsolidationAndPresentationOfFinancialStatementsLineItems" xlink:type="locator" xlink:label="astc_OrganizationConsolidationAndPresentationOfFinancialStatementsLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="astc_OrganizationConsolidationAndPresentationOfFinancialStatementsLineItems" xlink:to="astc_OrganizationConsolidationAndPresentationOfFinancialStatementsTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="astc_OrganizationConsolidationAndPresentationOfFinancialStatementsTable" xlink:to="us-gaap_StatementEquityComponentsAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="astc_OrganizationConsolidationAndPresentationOfFinancialStatementsTable" xlink:to="us-gaap_TypeOfArrangementAxis" order="10120.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember_2" order="10130.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" order="10150.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="astc_PublicOfferingPurchaseAgreementMember" order="10170.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="astc_RegisteredOfferingPurchaseAgreementMember" order="10190.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="astc_OrganizationConsolidationAndPresentationOfFinancialStatementsTable" xlink:to="us-gaap_SubsequentEventTypeAxis" order="10200.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain" order="10230.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventTypeDomain" xlink:to="us-gaap_SubsequentEventMember" order="10250.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_OrganizationConsolidationAndPresentationOfFinancialStatementsLineItems" xlink:to="us-gaap_ProceedsFromIssuanceOrSaleOfEquity" order="10300.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureLeasesAdditionalInformationDetails" xlink:href="astc-20200930.xsd#Role_DisclosureLeasesAdditionalInformationDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureLeasesAdditionalInformationDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_FinanceLeasePrincipalPayments" xlink:type="locator" xlink:label="us-gaap_FinanceLeasePrincipalPayments" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_OperatingLeasePayments" xlink:type="locator" xlink:label="us-gaap_OperatingLeasePayments" />
    <link:loc xlink:href="astc-20200930.xsd#astc_RemainingAreaOfLand" xlink:type="locator" xlink:label="astc_RemainingAreaOfLand" />
    <link:loc xlink:href="astc-20200930.xsd#astc_OriginalLeaseAreaOfLandRemoved" xlink:type="locator" xlink:label="astc_OriginalLeaseAreaOfLandRemoved" />
    <link:loc xlink:href="astc-20200930.xsd#astc_AreaOfLandRightOfFirstRefusalExercised" xlink:type="locator" xlink:label="astc_AreaOfLandRightOfFirstRefusalExercised" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LesseeOperatingLeaseTermOfContract" xlink:type="locator" xlink:label="us-gaap_LesseeOperatingLeaseTermOfContract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LesseeOperatingLeaseRenewalTerm" xlink:type="locator" xlink:label="us-gaap_LesseeOperatingLeaseRenewalTerm" />
    <link:loc xlink:href="astc-20200930.xsd#astc_NumberOfRenewalTerms" xlink:type="locator" xlink:label="astc_NumberOfRenewalTerms" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LesseeOperatingLeaseExistenceOfOptionToExtend" xlink:type="locator" xlink:label="us-gaap_LesseeOperatingLeaseExistenceOfOptionToExtend" />
    <link:loc xlink:href="astc-20200930.xsd#astc_DecreaseInOperatingLeaseLiability" xlink:type="locator" xlink:label="astc_DecreaseInOperatingLeaseLiability" />
    <link:loc xlink:href="astc-20200930.xsd#astc_DecreaseInOperatingLeaseRightOfUseAsset" xlink:type="locator" xlink:label="astc_DecreaseInOperatingLeaseRightOfUseAsset" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LeaseExpirationDate1" xlink:type="locator" xlink:label="us-gaap_LeaseExpirationDate1" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AreaOfLand" xlink:type="locator" xlink:label="us-gaap_AreaOfLand" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RetainedEarningsAccumulatedDeficit" xlink:type="locator" xlink:label="us-gaap_RetainedEarningsAccumulatedDeficit" />
    <link:loc xlink:href="astc-20200930.xsd#astc_ResearchAndDevelopmentAndProductionFacilityMember" xlink:type="locator" xlink:label="astc_ResearchAndDevelopmentAndProductionFacilityMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="locator" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="locator" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="locator" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis" />
    <link:loc xlink:href="astc-20200930.xsd#astc_FirstDetectCorporationMember" xlink:type="locator" xlink:label="astc_FirstDetectCorporationMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RelatedPartyDomain" xlink:type="locator" xlink:label="us-gaap_RelatedPartyDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RelatedPartyDomain" xlink:type="locator" xlink:label="us-gaap_RelatedPartyDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:type="locator" xlink:label="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" />
    <link:loc xlink:href="astc-20200930.xsd#astc_WebsterTexasMember" xlink:type="locator" xlink:label="astc_WebsterTexasMember" />
    <link:loc xlink:href="astc-20200930.xsd#astc_AustinTexasMember" xlink:type="locator" xlink:label="astc_AustinTexasMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_SegmentGeographicalDomain" xlink:type="locator" xlink:label="srt_SegmentGeographicalDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_SegmentGeographicalDomain" xlink:type="locator" xlink:label="srt_SegmentGeographicalDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_StatementGeographicalAxis" xlink:type="locator" xlink:label="srt_StatementGeographicalAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AccountingStandardsUpdate201602Member" xlink:type="locator" xlink:label="us-gaap_AccountingStandardsUpdate201602Member" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_TypeOfAdoptionMember" xlink:type="locator" xlink:label="us-gaap_TypeOfAdoptionMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_TypeOfAdoptionMember" xlink:type="locator" xlink:label="us-gaap_TypeOfAdoptionMember_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AdjustmentsForNewAccountingPronouncementsAxis" xlink:type="locator" xlink:label="us-gaap_AdjustmentsForNewAccountingPronouncementsAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_CumulativeEffectPeriodOfAdoptionAdjustmentMember" xlink:type="locator" xlink:label="srt_CumulativeEffectPeriodOfAdoptionAdjustmentMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_CumulativeEffectPeriodOfAdoptionDomain" xlink:type="locator" xlink:label="srt_CumulativeEffectPeriodOfAdoptionDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_CumulativeEffectPeriodOfAdoptionAxis" xlink:type="locator" xlink:label="srt_CumulativeEffectPeriodOfAdoptionAxis" />
    <link:loc xlink:href="astc-20200930.xsd#astc_LeasesTable" xlink:type="locator" xlink:label="astc_LeasesTable" />
    <link:loc xlink:href="astc-20200930.xsd#astc_LeasesLineItems" xlink:type="locator" xlink:label="astc_LeasesLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="astc_LeasesLineItems" xlink:to="astc_LeasesTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="astc_LeasesTable" xlink:to="srt_CumulativeEffectPeriodOfAdoptionAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_CumulativeEffectPeriodOfAdoptionAxis" xlink:to="srt_CumulativeEffectPeriodOfAdoptionDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_CumulativeEffectPeriodOfAdoptionDomain" xlink:to="srt_CumulativeEffectPeriodOfAdoptionAdjustmentMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="astc_LeasesTable" xlink:to="us-gaap_AdjustmentsForNewAccountingPronouncementsAxis" order="10100.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AdjustmentsForNewAccountingPronouncementsAxis" xlink:to="us-gaap_TypeOfAdoptionMember_2" order="10110.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AdjustmentsForNewAccountingPronouncementsAxis" xlink:to="us-gaap_TypeOfAdoptionMember" order="10130.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TypeOfAdoptionMember" xlink:to="us-gaap_AccountingStandardsUpdate201602Member" order="10150.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="astc_LeasesTable" xlink:to="srt_StatementGeographicalAxis" order="10160.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain_2" order="10170.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain" order="10190.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_SegmentGeographicalDomain" xlink:to="astc_AustinTexasMember" order="10210.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_SegmentGeographicalDomain" xlink:to="astc_WebsterTexasMember" order="10330.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="astc_LeasesTable" xlink:to="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" order="10380.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:to="us-gaap_RelatedPartyDomain_2" order="10390.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:to="us-gaap_RelatedPartyDomain" order="10410.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain" xlink:to="astc_FirstDetectCorporationMember" order="10430.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="astc_LeasesTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" order="10480.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain_2" order="10490.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" order="10510.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="astc_ResearchAndDevelopmentAndProductionFacilityMember" order="10530.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_LeasesLineItems" xlink:to="us-gaap_RetainedEarningsAccumulatedDeficit" order="10560.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_LeasesLineItems" xlink:to="us-gaap_AreaOfLand" order="10610.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_LeasesLineItems" xlink:to="us-gaap_LeaseExpirationDate1" order="10650.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_LeasesLineItems" xlink:to="astc_DecreaseInOperatingLeaseRightOfUseAsset" order="10690.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_LeasesLineItems" xlink:to="astc_DecreaseInOperatingLeaseLiability" order="10730.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_LeasesLineItems" xlink:to="us-gaap_LesseeOperatingLeaseExistenceOfOptionToExtend" order="11000.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_LeasesLineItems" xlink:to="astc_NumberOfRenewalTerms" order="11100.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_LeasesLineItems" xlink:to="us-gaap_LesseeOperatingLeaseRenewalTerm" order="11200.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_LeasesLineItems" xlink:to="us-gaap_LesseeOperatingLeaseTermOfContract" order="11300.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_LeasesLineItems" xlink:to="astc_AreaOfLandRightOfFirstRefusalExercised" order="11400.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_LeasesLineItems" xlink:to="astc_OriginalLeaseAreaOfLandRemoved" order="11800.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_LeasesLineItems" xlink:to="astc_RemainingAreaOfLand" order="11900.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_LeasesLineItems" xlink:to="us-gaap_OperatingLeasePayments" order="12030.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="astc_LeasesLineItems" xlink:to="us-gaap_FinanceLeasePrincipalPayments" order="12070.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosurePropertyAndEquipmentScheduleOfPropertyAndEquipmentNetDetails" xlink:href="astc-20200930.xsd#Role_DisclosurePropertyAndEquipmentScheduleOfPropertyAndEquipmentNetDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosurePropertyAndEquipmentScheduleOfPropertyAndEquipmentNetDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PropertyPlantAndEquipmentNet" xlink:type="locator" xlink:label="us-gaap_PropertyPlantAndEquipmentNet" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AssetsHeldForSaleNotPartOfDisposalGroup" xlink:type="locator" xlink:label="us-gaap_AssetsHeldForSaleNotPartOfDisposalGroup" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment" xlink:type="locator" xlink:label="us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PropertyPlantAndEquipmentGross" xlink:type="locator" xlink:label="us-gaap_PropertyPlantAndEquipmentGross" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ConstructionInProgressMember" xlink:type="locator" xlink:label="us-gaap_ConstructionInProgressMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SoftwareAndSoftwareDevelopmentCostsMember" xlink:type="locator" xlink:label="us-gaap_SoftwareAndSoftwareDevelopmentCostsMember" />
    <link:loc xlink:href="astc-20200930.xsd#astc_FurnitureFixturesEquipmentAndLeaseholdImprovementsMember" xlink:type="locator" xlink:label="astc_FurnitureFixturesEquipmentAndLeaseholdImprovementsMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:type="locator" xlink:label="us-gaap_PropertyPlantAndEquipmentTypeDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:type="locator" xlink:label="us-gaap_PropertyPlantAndEquipmentByTypeAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:type="locator" xlink:label="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PropertyPlantAndEquipmentLineItems" xlink:type="locator" xlink:label="us-gaap_PropertyPlantAndEquipmentLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" order="10040.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:to="us-gaap_PropertyPlantAndEquipmentByTypeAxis" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PropertyPlantAndEquipmentByTypeAxis" xlink:to="us-gaap_PropertyPlantAndEquipmentTypeDomain" order="10100.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="astc_FurnitureFixturesEquipmentAndLeaseholdImprovementsMember" order="10120.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_SoftwareAndSoftwareDevelopmentCostsMember" order="10140.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentTypeDomain" xlink:to="us-gaap_ConstructionInProgressMember" order="10160.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentGross" order="10190.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_AccumulatedDepreciationDepletionAndAmortizationPropertyPlantAndEquipment" order="10350.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_AssetsHeldForSaleNotPartOfDisposalGroup" order="10390.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentNet" order="10430.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosurePropertyAndEquipmentAdditionalInformationDetails" xlink:href="astc-20200930.xsd#Role_DisclosurePropertyAndEquipmentAdditionalInformationDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosurePropertyAndEquipmentAdditionalInformationDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_Depreciation" xlink:type="locator" xlink:label="us-gaap_Depreciation" />
    <link:loc xlink:href="astc-20200930.xsd#astc_WriteOffOfNetLeaseholdImprovementAssets" xlink:type="locator" xlink:label="astc_WriteOffOfNetLeaseholdImprovementAssets" />
    <link:loc xlink:href="astc-20200930.xsd#astc_AustinTexasMember" xlink:type="locator" xlink:label="astc_AustinTexasMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_SegmentGeographicalDomain" xlink:type="locator" xlink:label="srt_SegmentGeographicalDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_StatementGeographicalAxis" xlink:type="locator" xlink:label="srt_StatementGeographicalAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:type="locator" xlink:label="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PropertyPlantAndEquipmentLineItems" xlink:type="locator" xlink:label="us-gaap_PropertyPlantAndEquipmentLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfPropertyPlantAndEquipmentTable" xlink:to="srt_StatementGeographicalAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_StatementGeographicalAxis" xlink:to="srt_SegmentGeographicalDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_SegmentGeographicalDomain" xlink:to="astc_AustinTexasMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="astc_WriteOffOfNetLeaseholdImprovementAssets" order="10120.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PropertyPlantAndEquipmentLineItems" xlink:to="us-gaap_Depreciation" order="10170.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureStockholdersEquityDeficitAdditionalInformationDetails" xlink:href="astc-20200930.xsd#Role_DisclosureStockholdersEquityDeficitAdditionalInformationDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureStockholdersEquityDeficitAdditionalInformationDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ProceedsFromIssuanceOrSaleOfEquity" xlink:type="locator" xlink:label="us-gaap_ProceedsFromIssuanceOrSaleOfEquity" />
    <link:loc xlink:href="astc-20200930.xsd#astc_MaximumNumberOfDaysExtensionOnCompliancePeriod" xlink:type="locator" xlink:label="astc_MaximumNumberOfDaysExtensionOnCompliancePeriod" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockholdersEquity" xlink:type="locator" xlink:label="us-gaap_StockholdersEquity" />
    <link:loc xlink:href="astc-20200930.xsd#astc_DescriptionOfListingCompliance" xlink:type="locator" xlink:label="astc_DescriptionOfListingCompliance" />
    <link:loc xlink:href="astc-20200930.xsd#astc_AdditionalCommonStockSharesSold" xlink:type="locator" xlink:label="astc_AdditionalCommonStockSharesSold" />
    <link:loc xlink:href="astc-20200930.xsd#astc_SaleOfStockAverageStockPricePerShare" xlink:type="locator" xlink:label="astc_SaleOfStockAverageStockPricePerShare" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:type="locator" xlink:label="us-gaap_ProceedsFromIssuanceOfCommonStock" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="locator" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" />
    <link:loc xlink:href="astc-20200930.xsd#astc_PublicOfferingPurchaseAgreementMember" xlink:type="locator" xlink:label="astc_PublicOfferingPurchaseAgreementMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsequentEventMember" xlink:type="locator" xlink:label="us-gaap_SubsequentEventMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsequentEventTypeDomain" xlink:type="locator" xlink:label="us-gaap_SubsequentEventTypeDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsequentEventTypeAxis" xlink:type="locator" xlink:label="us-gaap_SubsequentEventTypeAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_MinimumMember" xlink:type="locator" xlink:label="srt_MinimumMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_MaximumMember" xlink:type="locator" xlink:label="srt_MaximumMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_RangeMember" xlink:type="locator" xlink:label="srt_RangeMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_RangeMember" xlink:type="locator" xlink:label="srt_RangeMember_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_RangeAxis" xlink:type="locator" xlink:label="srt_RangeAxis" />
    <link:loc xlink:href="astc-20200930.xsd#astc_MarketIssuanceSalesAgreementMember" xlink:type="locator" xlink:label="astc_MarketIssuanceSalesAgreementMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="locator" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_TypeOfArrangementAxis" xlink:type="locator" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockMember" xlink:type="locator" xlink:label="us-gaap_CommonStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EquityComponentDomain" xlink:type="locator" xlink:label="us-gaap_EquityComponentDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementEquityComponentsAxis" xlink:type="locator" xlink:label="us-gaap_StatementEquityComponentsAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ScheduleOfStockByClassTable" xlink:type="locator" xlink:label="us-gaap_ScheduleOfStockByClassTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfStockLineItems" xlink:type="locator" xlink:label="us-gaap_ClassOfStockLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_ScheduleOfStockByClassTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_StatementEquityComponentsAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_TypeOfArrangementAxis" order="10160.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" order="10190.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="astc_MarketIssuanceSalesAgreementMember" order="10210.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="srt_RangeAxis" order="10280.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember_2" order="10290.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" order="10310.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" order="10330.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" order="10350.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_SubsequentEventTypeAxis" order="10360.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain" order="10390.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventTypeDomain" xlink:to="us-gaap_SubsequentEventMember" order="10410.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="astc_PublicOfferingPurchaseAgreementMember" order="10530.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" order="10560.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_ProceedsFromIssuanceOfCommonStock" order="10580.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_SaleOfStockAverageStockPricePerShare" order="10600.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_AdditionalCommonStockSharesSold" order="10620.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_DescriptionOfListingCompliance" order="10670.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_StockholdersEquity" order="10710.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_MaximumNumberOfDaysExtensionOnCompliancePeriod" order="10790.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_ProceedsFromIssuanceOrSaleOfEquity" order="10830.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureStockholdersEquityDeficitSummaryOfCommonStockWarrantActivityDetails" xlink:href="astc-20200930.xsd#Role_DisclosureStockholdersEquityDeficitSummaryOfCommonStockWarrantActivityDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureStockholdersEquityDeficitSummaryOfCommonStockWarrantActivityDetails">
    <link:loc xlink:href="astc-20200930.xsd#astc_ClassOfWarrantsOrRightsOutstandingWeightedAverageRemainingContractualLife" xlink:type="locator" xlink:label="astc_ClassOfWarrantsOrRightsOutstandingWeightedAverageRemainingContractualLife" />
    <link:loc xlink:href="astc-20200930.xsd#astc_AggregateFairMarketValueAtIssuanceClassOfWarrantsOrRightsCanceledOrExpired" xlink:type="locator" xlink:label="astc_AggregateFairMarketValueAtIssuanceClassOfWarrantsOrRightsCanceledOrExpired" />
    <link:loc xlink:href="astc-20200930.xsd#astc_AggregateFairMarketValueAtIssuanceClassOfWarrantsOrRightsExercised" xlink:type="locator" xlink:label="astc_AggregateFairMarketValueAtIssuanceClassOfWarrantsOrRightsExercised" />
    <link:loc xlink:href="astc-20200930.xsd#astc_AggregateFairMarketValueAtIssuanceClassOfWarrantsOrRightsIssued" xlink:type="locator" xlink:label="astc_AggregateFairMarketValueAtIssuanceClassOfWarrantsOrRightsIssued" />
    <link:loc xlink:href="astc-20200930.xsd#astc_AggregateFairMarketValueAtIssuanceClassOfWarrantsOrRightsOutstanding" xlink:type="locator" xlink:label="astc_AggregateFairMarketValueAtIssuanceClassOfWarrantsOrRightsOutstanding" />
    <link:loc xlink:href="astc-20200930.xsd#astc_WeightedAverageExercisePriceClassOfWarrantsOrRightsCanceledOrExpired" xlink:type="locator" xlink:label="astc_WeightedAverageExercisePriceClassOfWarrantsOrRightsCanceledOrExpired" />
    <link:loc xlink:href="astc-20200930.xsd#astc_WeightedAverageExercisePriceClassOfWarrantsOrRightsExercised" xlink:type="locator" xlink:label="astc_WeightedAverageExercisePriceClassOfWarrantsOrRightsExercised" />
    <link:loc xlink:href="astc-20200930.xsd#astc_WeightedAverageExercisePriceClassOfWarrantsOrRightsIssued" xlink:type="locator" xlink:label="astc_WeightedAverageExercisePriceClassOfWarrantsOrRightsIssued" />
    <link:loc xlink:href="astc-20200930.xsd#astc_WeightedAverageExercisePriceClassOfWarrantsOrRightsOutstanding" xlink:type="locator" xlink:label="astc_WeightedAverageExercisePriceClassOfWarrantsOrRightsOutstanding" />
    <link:loc xlink:href="astc-20200930.xsd#astc_ClassOfWarrantsOrRightsExpiredDuringPeriod" xlink:type="locator" xlink:label="astc_ClassOfWarrantsOrRightsExpiredDuringPeriod" />
    <link:loc xlink:href="astc-20200930.xsd#astc_ClassOfWarrantsOrRightsExercisedDuringPeriod" xlink:type="locator" xlink:label="astc_ClassOfWarrantsOrRightsExercisedDuringPeriod" />
    <link:loc xlink:href="astc-20200930.xsd#astc_ClassOfWarrantsOrRightsIssuedDuringPeriod" xlink:type="locator" xlink:label="astc_ClassOfWarrantsOrRightsIssuedDuringPeriod" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfWarrantOrRightOutstanding" xlink:type="locator" xlink:label="us-gaap_ClassOfWarrantOrRightOutstanding" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_WarrantMember" xlink:type="locator" xlink:label="us-gaap_WarrantMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EquityComponentDomain" xlink:type="locator" xlink:label="us-gaap_EquityComponentDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementEquityComponentsAxis" xlink:type="locator" xlink:label="us-gaap_StatementEquityComponentsAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ScheduleOfStockByClassTable" xlink:type="locator" xlink:label="us-gaap_ScheduleOfStockByClassTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfStockLineItems" xlink:type="locator" xlink:label="us-gaap_ClassOfStockLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_ScheduleOfStockByClassTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_StatementEquityComponentsAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_WarrantMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightOutstanding" order="10300.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_ClassOfWarrantsOrRightsIssuedDuringPeriod" order="10320.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_ClassOfWarrantsOrRightsExercisedDuringPeriod" order="10340.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_ClassOfWarrantsOrRightsExpiredDuringPeriod" order="10360.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_WeightedAverageExercisePriceClassOfWarrantsOrRightsOutstanding" order="10430.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_WeightedAverageExercisePriceClassOfWarrantsOrRightsIssued" order="10450.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_WeightedAverageExercisePriceClassOfWarrantsOrRightsExercised" order="10470.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_WeightedAverageExercisePriceClassOfWarrantsOrRightsCanceledOrExpired" order="10490.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_AggregateFairMarketValueAtIssuanceClassOfWarrantsOrRightsOutstanding" order="10560.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_AggregateFairMarketValueAtIssuanceClassOfWarrantsOrRightsIssued" order="10580.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_AggregateFairMarketValueAtIssuanceClassOfWarrantsOrRightsExercised" order="10600.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_AggregateFairMarketValueAtIssuanceClassOfWarrantsOrRightsCanceledOrExpired" order="10620.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_ClassOfWarrantsOrRightsOutstandingWeightedAverageRemainingContractualLife" order="10690.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureStockholdersEquityDeficitScheduleOfWarrantsOutstandingDetails" xlink:href="astc-20200930.xsd#Role_DisclosureStockholdersEquityDeficitScheduleOfWarrantsOutstandingDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureStockholdersEquityDeficitScheduleOfWarrantsOutstandingDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfWarrantOrRightOutstanding" xlink:type="locator" xlink:label="us-gaap_ClassOfWarrantOrRightOutstanding" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_WarrantsAndRightsOutstandingMaturityDate" xlink:type="locator" xlink:label="us-gaap_WarrantsAndRightsOutstandingMaturityDate" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="locator" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" />
    <link:loc xlink:href="astc-20200930.xsd#astc_WarrantsAndRightsClassification" xlink:type="locator" xlink:label="astc_WarrantsAndRightsClassification" />
    <link:loc xlink:href="astc-20200930.xsd#astc_WarrantAndRightsOutstandingIssueDate" xlink:type="locator" xlink:label="astc_WarrantAndRightsOutstandingIssueDate" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_WarrantMember" xlink:type="locator" xlink:label="us-gaap_WarrantMember" />
    <link:loc xlink:href="astc-20200930.xsd#astc_WarrantsIssuedOnMarchThirtyTwoThousandTwentyMember" xlink:type="locator" xlink:label="astc_WarrantsIssuedOnMarchThirtyTwoThousandTwentyMember" />
    <link:loc xlink:href="astc-20200930.xsd#astc_WarrantsIssuedOnMarchTwentySixTwoThousandTwentyMember" xlink:type="locator" xlink:label="astc_WarrantsIssuedOnMarchTwentySixTwoThousandTwentyMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EquityComponentDomain" xlink:type="locator" xlink:label="us-gaap_EquityComponentDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementEquityComponentsAxis" xlink:type="locator" xlink:label="us-gaap_StatementEquityComponentsAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ScheduleOfStockByClassTable" xlink:type="locator" xlink:label="us-gaap_ScheduleOfStockByClassTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfStockLineItems" xlink:type="locator" xlink:label="us-gaap_ClassOfStockLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_ScheduleOfStockByClassTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_StatementEquityComponentsAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="astc_WarrantsIssuedOnMarchTwentySixTwoThousandTwentyMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="astc_WarrantsIssuedOnMarchThirtyTwoThousandTwentyMember" order="10110.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_WarrantMember" order="10130.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_WarrantAndRightsOutstandingIssueDate" order="10160.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="astc_WarrantsAndRightsClassification" order="10200.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" order="10240.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_WarrantsAndRightsOutstandingMaturityDate" order="10280.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightOutstanding" order="10320.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureDebtAdditionalInformationDetails" xlink:href="astc-20200930.xsd#Role_DisclosureDebtAdditionalInformationDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureDebtAdditionalInformationDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_InterestExpense" xlink:type="locator" xlink:label="us-gaap_InterestExpense" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DebtInstrumentPaymentTerms" xlink:type="locator" xlink:label="us-gaap_DebtInstrumentPaymentTerms" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DebtInstrumentFrequencyOfPeriodicPayment" xlink:type="locator" xlink:label="us-gaap_DebtInstrumentFrequencyOfPeriodicPayment" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ProceedsFromNotesPayable" xlink:type="locator" xlink:label="us-gaap_ProceedsFromNotesPayable" />
    <link:loc xlink:href="astc-20200930.xsd#astc_DebtInstrumentExtendedMaturityDate" xlink:type="locator" xlink:label="astc_DebtInstrumentExtendedMaturityDate" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DebtInstrumentMaturityDate" xlink:type="locator" xlink:label="us-gaap_DebtInstrumentMaturityDate" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DebtInstrumentInterestRateStatedPercentage" xlink:type="locator" xlink:label="us-gaap_DebtInstrumentInterestRateStatedPercentage" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DebtInstrumentFaceAmount" xlink:type="locator" xlink:label="us-gaap_DebtInstrumentFaceAmount" />
    <link:loc xlink:href="astc-20200930.xsd#astc_PaycheckProtectionProgramPromissoryNoteMember" xlink:type="locator" xlink:label="astc_PaycheckProtectionProgramPromissoryNoteMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PrivatePlacementMember" xlink:type="locator" xlink:label="us-gaap_PrivatePlacementMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:type="locator" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" />
    <link:loc xlink:href="astc-20200930.xsd#astc_SecuredPromissoryNoteMember" xlink:type="locator" xlink:label="astc_SecuredPromissoryNoteMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DebtInstrumentNameDomain" xlink:type="locator" xlink:label="us-gaap_DebtInstrumentNameDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DebtInstrumentNameDomain" xlink:type="locator" xlink:label="us-gaap_DebtInstrumentNameDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DebtInstrumentAxis" xlink:type="locator" xlink:label="us-gaap_DebtInstrumentAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DebtInstrumentTable" xlink:type="locator" xlink:label="us-gaap_DebtInstrumentTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DebtInstrumentLineItems" xlink:type="locator" xlink:label="us-gaap_DebtInstrumentLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="us-gaap_DebtInstrumentAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain_2" order="10050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="astc_SecuredPromissoryNoteMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_DebtInstrumentTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" order="10140.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain_2" order="10150.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" order="10170.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="us-gaap_PrivatePlacementMember" order="10190.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="astc_PaycheckProtectionProgramPromissoryNoteMember" order="10510.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentFaceAmount" order="10540.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentInterestRateStatedPercentage" order="10560.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentMaturityDate" order="10580.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="astc_DebtInstrumentExtendedMaturityDate" order="10700.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_ProceedsFromNotesPayable" order="10750.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentFrequencyOfPeriodicPayment" order="10810.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_DebtInstrumentPaymentTerms" order="10830.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentLineItems" xlink:to="us-gaap_InterestExpense" order="10850.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureCommonStockCompensationAdditionalInformationDetails" xlink:href="astc-20200930.xsd#Role_DisclosureCommonStockCompensationAdditionalInformationDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureCommonStockCompensationAdditionalInformationDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions" xlink:type="locator" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:type="locator" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" xlink:type="locator" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions" xlink:type="locator" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableIntrinsicValue1" xlink:type="locator" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableIntrinsicValue1" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RestrictedStockMember" xlink:type="locator" xlink:label="us-gaap_RestrictedStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EmployeeStockOptionMember" xlink:type="locator" xlink:label="us-gaap_EmployeeStockOptionMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AwardTypeAxis" xlink:type="locator" xlink:label="us-gaap_AwardTypeAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="locator" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_2" order="10050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockMember" order="10150.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableIntrinsicValue1" order="10220.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions" order="10240.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" order="10260.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" order="10310.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions" order="10420.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureCommonStockCompensationScheduleOfStockOptionsOutstandingDetails" xlink:href="astc-20200930.xsd#Role_DisclosureCommonStockCompensationScheduleOfStockOptionsOutstandingDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureCommonStockCompensationScheduleOfStockOptionsOutstandingDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeExercisableOptionsWeightedAverageExercisePrice1" xlink:type="locator" xlink:label="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeExercisableOptionsWeightedAverageExercisePrice1" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfExercisableOptions" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfExercisableOptions" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageExercisePriceBeginningBalance1" xlink:type="locator" xlink:label="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageExercisePriceBeginningBalance1" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageRemainingContractualTerm2" xlink:type="locator" xlink:label="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageRemainingContractualTerm2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfOutstandingOptions" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfOutstandingOptions" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeUpperRangeLimit" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeUpperRangeLimit" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLowerRangeLimit" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLowerRangeLimit" />
    <link:loc xlink:href="astc-20200930.xsd#astc_ExercisePriceRangeThreeMember" xlink:type="locator" xlink:label="astc_ExercisePriceRangeThreeMember" />
    <link:loc xlink:href="astc-20200930.xsd#astc_ExercisePriceRangeTwoMember" xlink:type="locator" xlink:label="astc_ExercisePriceRangeTwoMember" />
    <link:loc xlink:href="astc-20200930.xsd#astc_ExercisePriceRangeOneMember" xlink:type="locator" xlink:label="astc_ExercisePriceRangeOneMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeAxis" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeTable" xlink:type="locator" xlink:label="us-gaap_ScheduleOfShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLineItems" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeTable" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeAxis" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain_2" order="10050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeAxis" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:to="astc_ExercisePriceRangeOneMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:to="astc_ExercisePriceRangeTwoMember" order="10110.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:to="astc_ExercisePriceRangeThreeMember" order="10130.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLineItems" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLowerRangeLimit" order="10160.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLineItems" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeUpperRangeLimit" order="10260.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLineItems" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfOutstandingOptions" order="10360.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLineItems" xlink:to="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageRemainingContractualTerm2" order="10460.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLineItems" xlink:to="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageExercisePriceBeginningBalance1" order="10560.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLineItems" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfExercisableOptions" order="10660.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLineItems" xlink:to="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeExercisableOptionsWeightedAverageExercisePrice1" order="10760.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureCommonStockCompensationScheduleOfRestrictedStockActivityDetails" xlink:href="astc-20200930.xsd#Role_DisclosureCommonStockCompensationScheduleOfRestrictedStockActivityDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureCommonStockCompensationScheduleOfRestrictedStockActivityDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RestrictedStockMember" xlink:type="locator" xlink:label="us-gaap_RestrictedStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AwardTypeAxis" xlink:type="locator" xlink:label="us-gaap_AwardTypeAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="locator" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" order="10180.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" order="10300.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriod" order="10360.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" order="10420.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeitedInPeriod" order="10480.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" order="10630.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" order="10750.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" order="10810.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" order="10870.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValueRollForward" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsForfeituresWeightedAverageGrantDateFairValue" order="10930.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureIncomeTaxesAdditionalInformationDetails" xlink:href="astc-20200930.xsd#Role_DisclosureIncomeTaxesAdditionalInformationDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureIncomeTaxesAdditionalInformationDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_UnrecognizedTaxBenefitsPeriodIncreaseDecrease" xlink:type="locator" xlink:label="us-gaap_UnrecognizedTaxBenefitsPeriodIncreaseDecrease" />
    <link:loc xlink:href="astc-20200930.xsd#astc_RefundableAlternativeMinimumTaxCredits" xlink:type="locator" xlink:label="astc_RefundableAlternativeMinimumTaxCredits" />
    <link:loc xlink:href="astc-20200930.xsd#astc_PercentageOfTaxCreditCarryForwardsAlternativeMinimumTax" xlink:type="locator" xlink:label="astc_PercentageOfTaxCreditCarryForwardsAlternativeMinimumTax" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate" xlink:type="locator" xlink:label="us-gaap_EffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EffectiveIncomeTaxRateContinuingOperations" xlink:type="locator" xlink:label="us-gaap_EffectiveIncomeTaxRateContinuingOperations" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" xlink:type="locator" xlink:label="us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_MinimumMember" xlink:type="locator" xlink:label="srt_MinimumMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_MaximumMember" xlink:type="locator" xlink:label="srt_MaximumMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_RangeMember" xlink:type="locator" xlink:label="srt_RangeMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_RangeAxis" xlink:type="locator" xlink:label="srt_RangeAxis" />
    <link:loc xlink:href="astc-20200930.xsd#astc_CoronavirusAidReliefAndEconomicSecurityMember" xlink:type="locator" xlink:label="astc_CoronavirusAidReliefAndEconomicSecurityMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_UnusualOrInfrequentItemDomain" xlink:type="locator" xlink:label="us-gaap_UnusualOrInfrequentItemDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_UnusualOrInfrequentItemDomain" xlink:type="locator" xlink:label="us-gaap_UnusualOrInfrequentItemDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_UnusualOrInfrequentItemAxis" xlink:type="locator" xlink:label="us-gaap_UnusualOrInfrequentItemAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_FederalIncomeTaxNoteTable" xlink:type="locator" xlink:label="us-gaap_FederalIncomeTaxNoteTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_InvestmentsOwnedFederalIncomeTaxNoteLineItems" xlink:type="locator" xlink:label="us-gaap_InvestmentsOwnedFederalIncomeTaxNoteLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_InvestmentsOwnedFederalIncomeTaxNoteLineItems" xlink:to="us-gaap_FederalIncomeTaxNoteTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FederalIncomeTaxNoteTable" xlink:to="us-gaap_UnusualOrInfrequentItemAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_UnusualOrInfrequentItemAxis" xlink:to="us-gaap_UnusualOrInfrequentItemDomain_2" order="10050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_UnusualOrInfrequentItemAxis" xlink:to="us-gaap_UnusualOrInfrequentItemDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_UnusualOrInfrequentItemDomain" xlink:to="astc_CoronavirusAidReliefAndEconomicSecurityMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FederalIncomeTaxNoteTable" xlink:to="srt_RangeAxis" order="10120.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" order="10150.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" order="10170.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" order="10190.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_InvestmentsOwnedFederalIncomeTaxNoteLineItems" xlink:to="us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" order="10220.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_InvestmentsOwnedFederalIncomeTaxNoteLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateContinuingOperations" order="10260.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_InvestmentsOwnedFederalIncomeTaxNoteLineItems" xlink:to="us-gaap_EffectiveIncomeTaxRateReconciliationAtFederalStatutoryIncomeTaxRate" order="10330.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_InvestmentsOwnedFederalIncomeTaxNoteLineItems" xlink:to="astc_PercentageOfTaxCreditCarryForwardsAlternativeMinimumTax" order="10390.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_InvestmentsOwnedFederalIncomeTaxNoteLineItems" xlink:to="astc_RefundableAlternativeMinimumTaxCredits" order="10450.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_InvestmentsOwnedFederalIncomeTaxNoteLineItems" xlink:to="us-gaap_UnrecognizedTaxBenefitsPeriodIncreaseDecrease" order="10540.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureSegmentInformationScheduleOfSegmentReportingInformationBySegmentDetails" xlink:href="astc-20200930.xsd#Role_DisclosureSegmentInformationScheduleOfSegmentReportingInformationBySegmentDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureSegmentInformationScheduleOfSegmentReportingInformationBySegmentDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_Assets" xlink:type="locator" xlink:label="us-gaap_Assets" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PaymentsToAcquirePropertyPlantAndEquipment" xlink:type="locator" xlink:label="us-gaap_PaymentsToAcquirePropertyPlantAndEquipment" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PropertyPlantAndEquipmentNet" xlink:type="locator" xlink:label="us-gaap_PropertyPlantAndEquipmentNet" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" xlink:type="locator" xlink:label="us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_Depreciation" xlink:type="locator" xlink:label="us-gaap_Depreciation" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_Revenues" xlink:type="locator" xlink:label="us-gaap_Revenues" />
    <link:loc xlink:href="astc-20200930.xsd#astc_AgLabMember" xlink:type="locator" xlink:label="astc_AgLabMember" />
    <link:loc xlink:href="astc-20200930.xsd#astc_FirstDetectCorporationMember" xlink:type="locator" xlink:label="astc_FirstDetectCorporationMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SegmentDomain" xlink:type="locator" xlink:label="us-gaap_SegmentDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SegmentDomain" xlink:type="locator" xlink:label="us-gaap_SegmentDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementBusinessSegmentsAxis" xlink:type="locator" xlink:label="us-gaap_StatementBusinessSegmentsAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:type="locator" xlink:label="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SegmentReportingInformationLineItems" xlink:type="locator" xlink:label="us-gaap_SegmentReportingInformationLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfSegmentReportingInformationBySegmentTable" xlink:to="us-gaap_StatementBusinessSegmentsAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementBusinessSegmentsAxis" xlink:to="us-gaap_SegmentDomain_2" order="10050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementBusinessSegmentsAxis" xlink:to="us-gaap_SegmentDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentDomain" xlink:to="astc_FirstDetectCorporationMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentDomain" xlink:to="astc_AgLabMember" order="10110.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_Revenues" order="10220.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_Depreciation" order="10320.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_IncomeLossFromContinuingOperationsBeforeIncomeTaxesExtraordinaryItemsNoncontrollingInterest" order="10420.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_PropertyPlantAndEquipmentNet" order="10850.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_PaymentsToAcquirePropertyPlantAndEquipment" order="10950.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SegmentReportingInformationLineItems" xlink:to="us-gaap_Assets" order="11050.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureSubsequentEventsAdditionalInformationDetails" xlink:href="astc-20200930.xsd#Role_DisclosureSubsequentEventsAdditionalInformationDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://www.astrotechcorp.com/20200930/taxonomy/role/DisclosureSubsequentEventsAdditionalInformationDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_WarrantsAndRightsOutstandingMaturityDate" xlink:type="locator" xlink:label="us-gaap_WarrantsAndRightsOutstandingMaturityDate" />
    <link:loc xlink:href="astc-20200930.xsd#astc_PercentageOfPerShareOfferingPriceOfShares" xlink:type="locator" xlink:label="astc_PercentageOfPerShareOfferingPriceOfShares" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" xlink:type="locator" xlink:label="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" />
    <link:loc xlink:href="astc-20200930.xsd#astc_PercentageOfSharesSoldInOffering" xlink:type="locator" xlink:label="astc_PercentageOfSharesSoldInOffering" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfWarrantOrRightOutstanding" xlink:type="locator" xlink:label="us-gaap_ClassOfWarrantOrRightOutstanding" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ProceedsFromIssuanceOrSaleOfEquity" xlink:type="locator" xlink:label="us-gaap_ProceedsFromIssuanceOrSaleOfEquity" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SharesIssuedPricePerShare" xlink:type="locator" xlink:label="us-gaap_SharesIssuedPricePerShare" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:type="locator" xlink:label="us-gaap_CommonStockParOrStatedValuePerShare" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="locator" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CashReserveDepositRequiredAndMade" xlink:type="locator" xlink:label="us-gaap_CashReserveDepositRequiredAndMade" />
    <link:loc xlink:href="astc-20200930.xsd#astc_PlacementAgentMember" xlink:type="locator" xlink:label="astc_PlacementAgentMember" />
    <link:loc xlink:href="astc-20200930.xsd#astc_SecondPurchaseAgreementMember" xlink:type="locator" xlink:label="astc_SecondPurchaseAgreementMember" />
    <link:loc xlink:href="astc-20200930.xsd#astc_HCWainwrightCoLLCMember" xlink:type="locator" xlink:label="astc_HCWainwrightCoLLCMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RelatedPartyDomain" xlink:type="locator" xlink:label="us-gaap_RelatedPartyDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:type="locator" xlink:label="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_WarrantMember" xlink:type="locator" xlink:label="us-gaap_WarrantMember" />
    <link:loc xlink:href="astc-20200930.xsd#astc_PublicOfferingPurchaseAgreementMember" xlink:type="locator" xlink:label="astc_PublicOfferingPurchaseAgreementMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockMember" xlink:type="locator" xlink:label="us-gaap_CommonStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EquityComponentDomain" xlink:type="locator" xlink:label="us-gaap_EquityComponentDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementEquityComponentsAxis" xlink:type="locator" xlink:label="us-gaap_StatementEquityComponentsAxis" />
    <link:loc xlink:href="astc-20200930.xsd#astc_CommercialBankMember" xlink:type="locator" xlink:label="astc_CommercialBankMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LineOfCreditFacilityLenderDomain" xlink:type="locator" xlink:label="us-gaap_LineOfCreditFacilityLenderDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LineOfCreditFacilityLenderDomain" xlink:type="locator" xlink:label="us-gaap_LineOfCreditFacilityLenderDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LineOfCreditFacilityAxis" xlink:type="locator" xlink:label="us-gaap_LineOfCreditFacilityAxis" />
    <link:loc xlink:href="astc-20200930.xsd#astc_CashReserveAgreementMember" xlink:type="locator" xlink:label="astc_CashReserveAgreementMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:type="locator" xlink:label="us-gaap_ArrangementsAndNonarrangementTransactionsMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_TypeOfArrangementAxis" xlink:type="locator" xlink:label="us-gaap_TypeOfArrangementAxis" />
    <link:loc xlink:href="astc-20200930.xsd#astc_PaycheckProtectionProgramPromissoryNoteMember" xlink:type="locator" xlink:label="astc_PaycheckProtectionProgramPromissoryNoteMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DebtInstrumentNameDomain" xlink:type="locator" xlink:label="us-gaap_DebtInstrumentNameDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DebtInstrumentAxis" xlink:type="locator" xlink:label="us-gaap_DebtInstrumentAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsequentEventMember" xlink:type="locator" xlink:label="us-gaap_SubsequentEventMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsequentEventTypeDomain" xlink:type="locator" xlink:label="us-gaap_SubsequentEventTypeDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsequentEventTypeAxis" xlink:type="locator" xlink:label="us-gaap_SubsequentEventTypeAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsequentEventTable" xlink:type="locator" xlink:label="us-gaap_SubsequentEventTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsequentEventLineItems" xlink:type="locator" xlink:label="us-gaap_SubsequentEventLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_SubsequentEventLineItems" xlink:to="us-gaap_SubsequentEventTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_SubsequentEventTable" xlink:to="us-gaap_SubsequentEventTypeAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsequentEventTypeAxis" xlink:to="us-gaap_SubsequentEventTypeDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventTypeDomain" xlink:to="us-gaap_SubsequentEventMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_SubsequentEventTable" xlink:to="us-gaap_DebtInstrumentAxis" order="10100.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_DebtInstrumentAxis" xlink:to="us-gaap_DebtInstrumentNameDomain" order="10130.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_DebtInstrumentNameDomain" xlink:to="astc_PaycheckProtectionProgramPromissoryNoteMember" order="10150.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_SubsequentEventTable" xlink:to="us-gaap_TypeOfArrangementAxis" order="10160.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_TypeOfArrangementAxis" xlink:to="us-gaap_ArrangementsAndNonarrangementTransactionsMember" order="10190.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="astc_CashReserveAgreementMember" order="10210.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_SubsequentEventTable" xlink:to="us-gaap_LineOfCreditFacilityAxis" order="10220.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_LineOfCreditFacilityAxis" xlink:to="us-gaap_LineOfCreditFacilityLenderDomain_2" order="10230.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_LineOfCreditFacilityAxis" xlink:to="us-gaap_LineOfCreditFacilityLenderDomain" order="10250.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LineOfCreditFacilityLenderDomain" xlink:to="astc_CommercialBankMember" order="10270.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_SubsequentEventTable" xlink:to="us-gaap_StatementEquityComponentsAxis" order="10280.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" order="10310.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" order="10330.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="astc_PublicOfferingPurchaseAgreementMember" order="10430.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_WarrantMember" order="10810.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_SubsequentEventTable" xlink:to="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" order="11040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_RelatedPartyTransactionsByRelatedPartyAxis" xlink:to="us-gaap_RelatedPartyDomain" order="11070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain" xlink:to="astc_HCWainwrightCoLLCMember" order="11090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ArrangementsAndNonarrangementTransactionsMember" xlink:to="astc_SecondPurchaseAgreementMember" order="11310.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RelatedPartyDomain" xlink:to="astc_PlacementAgentMember" order="11930.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems" xlink:to="us-gaap_CashReserveDepositRequiredAndMade" order="12040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" order="12090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems" xlink:to="us-gaap_CommonStockParOrStatedValuePerShare" order="12110.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems" xlink:to="us-gaap_SharesIssuedPricePerShare" order="12130.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems" xlink:to="us-gaap_ProceedsFromIssuanceOrSaleOfEquity" order="12180.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightOutstanding" order="12230.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems" xlink:to="astc_PercentageOfSharesSoldInOffering" order="12250.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems" xlink:to="us-gaap_ClassOfWarrantOrRightExercisePriceOfWarrantsOrRights1" order="12270.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems" xlink:to="astc_PercentageOfPerShareOfferingPriceOfShares" order="12290.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SubsequentEventLineItems" xlink:to="us-gaap_WarrantsAndRightsOutstandingMaturityDate" order="12310.00" priority="2" use="optional" />
  </link:definitionLink>
</link:linkbase>