Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor, accounting for 50% of all cases. GBM patients have a five-year survival rate of merely 5.6% and a median overall survival of 14.6 months with the “Stupp” regimen, 20.9 months with tumor treatment fields (TTF, OptuneR) in patients who participated in clinical trials, and 11 months for all GBM patients prior to TTF use. Objective: Our group recently developed a brain cancer chip which generates tumor spheroids, and provides large-scale assessments on the response of tumor cells to various concentrations and combinations of drugs. This platform could optimize the use of tumor samples derived from GBM patients to provide valuable insight on the tumor growth and responses to drug therapies. To minimize any sample loss in vitro, we improved our brain cancer chip system by adding an additional laminar flow distribution layer, which reduces sample loss during cell seeding and prevents spheroids from escaping from the microwells. Methods: In this study, we cultured 3D spheroids from GBM cell lines and patient-derived GBM cells in vitro, and investigated the effect of the combination of Temozolomide and nuclear factor-κB inhibitor on tumor growth. Results: Our study revealed that these drugs have synergistic effects in inhibiting spheroid formation when used in combination. Conclusions: These results suggest that the brain cancer chip enables large-scale, inexpensive and sample-effective drug screening to 3D cancer tumors in vitro, and could be applied to related tissue engineering drug screening studies.