Drug permeability through cell monolayer is known to correlate well with in situ intestinal permeability and/or oral bioavailability. Several mammalian cell lines such as Caco-2, MDCK, MDCKII, and LLC-PK have been used to predict in vivo intestinal absorption of drugs. However, there are no well-characterized cell lines available representing the blood-brain barrier. In this chapter, the authors describe the primary culture of bovine brain microvessel endothelial cells (BMECs) lining the interface between the blood and the brain as the model for screening central nervous system (CNS) drug candidates. The culture procedures and measurement of permeability in BMEC can be applied to other model cell lines such as Caco-2, MDCK, and MDCKII cells.