This study evaluated the feasibility of using commercially available immortalized cell lines in building an in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Astrocytes-derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel biomimetic basement membrane for the endothelial cells to form tight junctions. Trans-Endothelial Electrical Resistance (TEER) and solute mass transport studies quantitatively evaluated the tight junction formation. Immuno-fluorescence microscopy and Western blot analysis qualitatively verified the expression of occludin, one of the tight junction proteins on the samples. Experimental data from a total of 13 experiments conclusively showed that the novel BBB in vitro co-culture model with aECM (CO + aECM) is promising in terms of establishing tight junction formation represented by TEER values, transport profiles, and tight junction protein expression when compared with traditional co-culture (CO) model setup or the endothelial cells cultured alone (EC). In vitro colorimetric sulforhodamine B (SRB) assay also revealed that the "CO + aECM" samples resulted in less cell loss on the basal sides of the insert membranes than traditional co-culture models. Our novel approach using immortalized cell lines with the addition of aECM was proven to be a feasible and repeatable alternative to the traditional BBB in vitro modeling.