Glioblastoma (GBM) is uniformly fatal with a 5-year survival rate of approximately 5%. Treatment options remain antiquated and ineffective. Most drugs fail to penetrate the blood brain barrier (BBB) and never reach the tumor. Among myriad challenges present with identifying novel therapeutics for GBM, not least is development of a high-throughput, in vitro model of an intact BBB. The BBB is selective and prevents most substances from crossing, providing both protection from toxins and simultaneously, the inability to get potential toxins (therapies) across and into a brain tumor. We have developed a reproducible, robust, and working model of primary human brain cells that, when correctly cultured, form tight junctions and an impermeant barrier. Ultimately, the model will be utilized to assess extracellular vesicle (EV), small molecule, and other therapeutic opportunities, providing the ability to devise and test novel therapies. Briefly, human primary astrocytes (HA), vascular pericytes (vP), and brain microvascular endothelial cells (EC) are cultured to form a spheroid. The spheroid core is composed of astrocytes and the ECs and vPs are layered to combine on the exterior to form tight junctions of an impermeant membrane. Various iterations of cell number, cell layering and the timing of each cells addition to the spheroid have been tried and optimized. Functionally, permeability assays using various sized (50 nm to 200 nm) fluorescently labeled nano plastics and FITC-dextran show an inability to cross the BBB-layer. Qualitatively, extensive immunocytochemical staining using tight junction markers ZO-1, S100-A10, Cloudin-5 and cell-specific markers (GFAP, CD31, PDGFR-beta) demonstrate that the relationship and arrangement of these cells produce and maintain tight junctions. Spheroids remain healthy, impermeant and functional for 10 days, providing a valid, time course for drug permeability assays. Future experiments will assess the limitations and size-selectivity of unique nanoparticles and extracellular vesicles loaded with novel therapeutic compounds.