Our newly developed drug delivery carrier, cationic bovine serum albumin (CBSA) conjugated with poly(ethyleneglycol)-poly(lactide) (PEG-PLA) nanoparticle (CBSA-NP), was designed for brain drug delivery. CBSA, as a brain specific targetor, was covalently conjugated with the maleimide function group at the distal of poly(ethyleneglycol) (PEG) surrounding the nanoparticles. To evaluate its blood-brain barrier (BBB) transcytosis and toxicity against the BBB endothelial tight junction, we have explored a method of coculture with brain capillary endothelial cells (BCECs) on the top of micro-porous membrane of cell culture insert and astrocytes on the bottom side. The permeability of 14C-labeled sucrose was determined. For the CBSA-NP transcytosis study, a lipophilic fluorescent probe, 6-coumarin, was incorporated into nanoparticles. The BBB permeability of CBSA-NP in vitro was calculated and compared with native bovine serum albumin (BSA) conjugated pegylated nanoparticles (BSA-NP). As the coculture model, the transendothelial electrical resistance reached up to 313