Introduction The efficiency of brain therapeutics is greatly hindered by the blood-brain barrier (BBB). BBB’s protective function, selective permeability, and dynamic functionality maintain the harmony between the brain and peripheral region. Thus, the design of any novel drug carrier system requires the complete study and investigation of BBB permeability, efflux transport, and the effect of associated cellular and non-vascular unit trafficking on BBB penetrability. The in vitro BBB models offer a most promising, and reliable mode of initial investigation of BBB permeability and associated factors as strong evidence for further preclinical and clinical investigation.