Patients　with　epidermal　growth　factor　receptor　(EGFR)　wild-type　non-small　cell　lung　cancer　(NSCLC)　often　show　primary　resistance　to　gefitinib　therapy.　It　is　thus　necessary　to　study　the　metabolism　of　gefitinib　in　NSCLC　cells　to　comprehensively　reveal　the　reasons　for　the　primary　resistance　of　tumors.　Herein,　we　develop　a　platform　for　studying　drug　metabolism　heterogeneity　based　on　single-cell　mass　spectrometry　(sDMH-scMS)　by　integrating　living-cell　electrolaunching　ionization　MS　(ILCEI-MS)　and　high-performance　liquid　chromatography-MS　(HPLC-MS)　analysis,　and　the　primary　resistance　of　NSCLC　cells　to　gefitinib　was　studied　using　this　platform.　The　ILCEI-MS　analysis　showed　that　approximately　11.9%　of　NSCLC　single　cells　contained　the　gefitinib　metabolite　M11　;　HPLC-MS　detection　diluted　the　intensity　of　M11　in　subpopulations　and　concealed　the　heterogeneity　of　drug　metabolism　in　tumor　single　cells.　The　intensity　of　gefitinib　in　EGFR　wild-type　A549　cells　was　markedly　lower　than　in　mutant　PC9　cells,　and　the　intensity　of　gefitinib　metabolites　was　significantly　higher　than　in　PC9　cells,　suggesting　that　the　primary　resistance　of　NSCLC　cells　is　related　to　gefitinib　metabolism.　Moreover,　the　combination　of　gefitinib　and　the　drug-metabolizing　enzyme　inhibitor　alpha-naphthoflavone　was　shown　to　overcome　the　primary　resistance　of　the　NSCLC　cells.　Overall,　the　results　of　this　study　are　expected　to　be　applicable　for　clinical　drug　resistance　diagnosis　and　treatment　at　the　single-cell　level.(c)　2023　Published　by　Elsevier　B.V.　on　behalf　of　Chinese　Chemical　Society　and　Institute　of　Materia　Medica,　Chinese　Academy　of　Medical　Sciences.