The three-dimensional (3D) organization of cells affects their mobility, proliferation, and overall response to treatment. Spheroids, organoids, and microfluidic chips are used in cancer research to reproduce in vitro the complex and dynamic malignant microenvironment. Herein, single- and double-channel microfluidic devices are used to mimic the spatial organization of brain tumors and investigate the therapeutic efficacy of molecular and nano anti-cancer agents. Human glioblastoma multiforme (U87-MG) cells were cultured into a Matrigel matrix embedded within the microfluidic devices and exposed to different doses of free docetaxel (DTXL), docetaxel-loaded spherical polymeric nanoparticles (DTXL-SPN), and the aromatic N-glucoside N-(fluorenylmethoxycarbonyl)-glucosamine-6-phosphate (Fmoc-Glc6P). We observed that in the single-channel microfluidic device, brain tumor cells are more susceptible to DTXL treatment as compared to conventional cell monolayers (50-fold lower IC50 values). In the double-channel device, the cytotoxicity of free DTXL and DTXL-SPN is comparable, but significantly lowered as compared to the single-channel configuration. Finally, the administration of 500 μM Fmoc-Glc6P in the double-channel microfluidic device shows a 50 % U87-MG cell survival after only 24 h, and no deleterious effect on human astrocytes over 72 h. Concluding, the proposed microfluidic chips can be used to reproduce the 3D complex spatial arrangement of solid tumors and to assess the anti-cancer efficacy of therapeutic compounds administrated in situ or systemically.