BACKGROUND Gemcitabine/cisplatin (GP) resistance displays a negative role in treating advanced and metastatic non-small cell lung cancer (NSCLC). Several studies found that the association existed between platelets and cancer antigen 125 (CA125) with anticancer drugs. But the exact correlation between GP resistance and platelet activation index remains poorly understood. MATERIAL AND METHODS Pre-chemotherapy platelet activation index and CA125 were retrospectively evaluated in 169 advanced and metastatic NSCLC patients. All variables were screened by chi-square test and then evaluated by log-rank test. Survival curves were generated by Kaplan-Meier analysis. Univariate and multivariate survival analysis were performed by using Cox proportional hazards model. RESULTS The overall rate of GP resistance for NSCLC patients was 72.19%. Mean platelet volume (MPV) and plateletcrit (PCT) are negative predictors of GP resistance adenocarcinoma [Odds ratio (OR): 5.81, 95% confidence interval (CI): 1.082-31.195, P=0.004] and squamous cell carcinoma (PCT: R: 3.517, 95% CI: 1.087-11.387, P=0.036), respectively. But both were an independent factor associated with overall survival (OS). Moreover, only CA125 was a dependent factor associated with OS for squamous cell carcinoma [OS: hazard ratio (HR): 1.741, 95% CI: 1.002-3.024, P=0.049; GP resistance: OR: 4.862, 95% CI: 1.437-16.448, P=0.011]. CONCLUSIONS Platelet activation index will be a potential marker for predicting GP resistance. Besides, CA125 ≥16.9 could be used as a potential marker for predicting GP resistance and OS, which was more sensitive than CA125 ≥35 for squamous cell carcinoma.