Studying　the　mechanisms　of　drug　antitumor　activity　at　the　single-cell　level　can　provide　information　about　the　responses　of　cell　subpopulations　to　drug　therapy,　which　is　essential　for　the　accurate　treatment　of　cancer.　Due　to　the　small　size　of　single　cells　and　the　low　contents　of　metabolites,　metabolomics-based　approaches　to　studying　the　mechanisms　of　drug　action　at　the　single-cell　level　are　lacking.　Herein,　we　develop　a　label-free　platform　for　studying　the　mechanisms　of　drug　action　based　on　single-cell　metabolomics　(sMDA-scM)　by　integrating　intact　living-cell　electro-launching　ionization　mass　spectrometry　(ILCEI-MS)　with　metabolomics　analysis.　Using　this　platform,　we　reveal　that　non-small-cell　lung　cancer　(NSCLC)　cells　treated　by　gefitinib　can　be　clustered　into　two　cell　subpopulations　with　different　metabolic　responses.　The　glutathione　metabolic　pathway　of　the　subpopulation　containing　14.4%　of　the　cells　is　not　significantly　affected　by　gefitinib,　exhibiting　certain　resistance　characteristics.　The　presence　of　these　cells　masked　the　judgment　of　whether　cysteine　and　methionine　metabolic　pathway　was　remarkably　influenced　in　the　analysis　of　overall　average　results,　revealing　the　heterogeneity　of　the　response　of　single　NSCLC　cells　to　gefitinib　treatment.　The　findings　provide　a　basis　for　evaluating　the　early　therapeutic　effects　of　clinical　medicines　and　insights　for　overcoming　drug　resistance　in　NSCLC　subpopulations.