The prognosis of malignant gliomas remains poor, despite the progress of surgery and radiotherapy. Chemotherapy has been shown to prolong an overall survival, but the benefits are still small. To overcome this situation, the optimal regimen of antineoplastic agents is required. In the present study, we investigated the effect of the association of five chemotherapeutic drugs, including ACNU, CBDCA, CDDP, VCR, and VP-16, on cell survival of U87, YKG1, A172, and U251 human glioma cell lines, using median-effect analysis. A synergistic effect was obtained by treatment involving the association of VP-16 with ACNU or CDDP among the combinations of two drugs, and the association of ACNU, CBDCA, and VP-16 in the combination of three drugs. This preclinical screening using median-effect analysis supports the design of clinical trials by indicating more effective combinations of antineoplastic agents for malignant gliomas.