Brain tumors are among the most heterogeneous tumors characterized. This heterogeneity drives resistance to single-drug therapies, making combination treatment a more promising approach to improve treatment outcomes. To optimize drug combinations and guide treatment development in this new era of combinatorial therapy, methods for quantifying synergistic, additive or antagonistic interactions between treatments are needed. In this chapter, we describe an in vitro assay to identify synergistic drug combinations in brain-tumor-derived cell cultures. As the gold-standard assay for the in vitro evaluation of drug combination efficacy, this protocol is a fast, inexpensive, and highly reproducible method for identifying and validating synergistic drug combinations.