Background: Selecting chemotherapy regimens guided by chemosensitivity tests can provide individualized therapies for cancer patients. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay is one in vitro assay which has become widely used to evaluate the sensitivity to anticancer agents. The aim of this study was to evaluate the clinical applicability and accuracy of MTS assay for predicting chemotherapeutic response in unresectable NSCLC patients. Methods: Cancer cells were isolated from malignant pleural effusions of patients by density gradient centrifugation, and their sensitivity to eight chemotherapeutic agents was examined by MTS assay and compared with clinical response. Results: A total of 37 patients participated in this study, and MTS assay produced results successfully in 34 patients (91.9%). The sensitivity rates ranged from 8.8% to 88.2%. Twenty-four of 34 patients who received chemotherapy were evaluated for in vitro-in vivo response analysis. The correlation between in vitro chemosensitivity result and in vivo response was highly significant (P=0.003), and the total predictive accuracy, sensitivity, specificity, positive predictive value, and negative predictive value for MTS assay were 87.5%, 94.1%, 71.4%, 88.9%, and 83.3%, respectively. The in vitro sensitivity for CDDP also showed a significant correlation with in vivo response (P=0.018, r=0.522). Conclusion: MTS assay is a preferable in vitro chemosensitivity assay that could be use to predict the response to chemotherapy and select the appropriate chemotherapy regimens for unresectable NSCLC patients, which could greatly improve therapeutic efficacy and reduce unnecessary adverse effects.