In vitro cell culture models of the blood–brain barrier (BBB) are important tools used to study cellular physiology and brain disease therapeutics. Although the number of model configurations is expanding across neuroscience laboratories, it is not clear that any have been effectively optimized. A sequential screening study to identify optimal primary mouse endothelial cell parameter set points, grown alone and in combination with common model enhancements, including co-culturing with primary mouse or rat astrocytes and addition of biochemical agents in the media, was performed. A range of endothelial cell-seeding densities (1–8 × 105 cells/cm2) and astrocyte-seeding densities (2–8 × 104 cells/cm2) were studied over seven days in the system, and three distinct media-feeding strategies were compared to optimize biochemical agent exposure time. Implementation of all optimal set points increased transendothelial electrical resistance by over 200% compared to an initial model and established a suitable in vitro model for brain disease application studies. These results demonstrate the importance of optimizing cell culture growth, which is the most important parameter in creating an in vitro BBB model as it directly relates the model to the in vivo arrangement.