Brain penetration is characterized by its extent and rate and is influenced by drug physicochemical properties, plasma exposure, plasma and brain protein binding and BBB permeability. This raises questions related to physiology, interspecies differences and in vitro/in vivo extrapolation. We herein discuss the use of in vitro human and animal BBB model as a tool to improve CNS compound selection. These cell-based BBB models are characterized by low paracellular permeation, well-developed tight junctions and functional efflux transporters. A study of twenty drugs shows similar compound ranking between rat and human models although with a 2-fold higher permeability in rat. cLogP < 5, PSA < 120 Å, MW < 450 were confirmed as essential for CNS drugs. An in vitro/in vivo correlation in rat (R² = 0.67; P = 2 × 10⁻⁴) was highlighted when in vitro permeability and efflux were considered together with plasma exposure and free fraction. The cell-based BBB model is suitable to optimize CNS-drug selection, to study interspecies differences and then to support human brain exposure prediction.