Acquired resistance is a major reason for poor clinical outcomes in cancer chemotherapy patients. The aim of this study was to determine the sensitivity to anticancer drugs and to identify the alterations of DNA repair and drug transporter in a model of primary culture obtained from pre- and post-platinum-based anticancer treatments in nine Thai gastric cancer patients. Ex vivo sensitivity to anti-cancer drugs (cisplatin, oxaliplatin, 5-fluorouracil (5-FU) and irinotecan) was analysed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of the drug transporter (multidrug resistance-associated protein 1 (MRP1), P-glycoprotein (P-gp)) and DNA repair (X-ray cross-complementing gene 1 (XRCC1) and excision repair cross-complementing 1 (ERCC1)) were examined by RT-PCR. The IC₅₀ to cisplatin and oxaliplatin of the cells obtained from gastric cancer patients after clinical drug treatments were administered to five patients (55.5%) revealed a significant increase when compared with prior treatments. The basal expression values of XRCC1, ERCC1 and MRP1 obtained from the treated patients were in correlation with those of IC₅₀. Ex vivo platinum drug treatment of the primary culture obtained from naïve patients over seven days also revealed a significant increase in MRP1 (7/9), XRCC1 (4/9) and ERCC1 (4/9). These observations have also been observed in the KATOIII cell line. Clinical treatment by platinum-based anti-cancer drug can develop acquired drug resistance in Thai gastric cancer patients through upregulation in the expression of drug transporter MRP1 and DNA repair XRCC1 and ERCC1. In cell culture model, cisplatin-resistant gastric cancer cell line KATOIII/diamminedichloroplatinum (KATOIII/DDP) significantly increased the expression level of these genes when compared to its parental cells (KATOIII).