The availability of good in vitro blood-brain barrier (BBB) models that closely mimic in vivo BBB features are essential for central nervous system (CNS) drug permeability screening and BBB functionality studies. Of the currently available monoculture primary BBB models, porcine brain endothelial cell models have the best barrier properties, which make them highly suitable for CNS drug permeability screening. In addition, they retain major BBB features such as BBB transporters, receptors, and enzymes and express BBB tight junctions. Therefore, porcine BBB models are also suitable for BBB functionality studies. This paper describes a procedure for extraction of brain microvessels from fresh porcine brains and the culture of pure primary porcine brain endothelial cells. In addition, techniques to improve culture purity and quality, and increase barrier tightness without using co-cultures are given. Using this method, a robust and reproducible in vitro BBB model can be established for CNS permeability screening and studying BBB functionality.