The blood-brain barrier (BBB) is an active interface between the circulation and the central nervous system (CNS) with a dual function: the barrier function restricts the transport from the blood to the brain of potentially toxic or harmful substances; the carrier function is responsible for the transport of nutrients to the brain and removal of metabolites. The BBB plays a crucial role in the clinical practice as well. On the one side there is a large number of neurological disorders including cerebral ischemia, brain trauma and tumors, neurodegenerative disorders, in which the permeability of the BBB is increased. On the other hand due to the relative impermeability of the barrier many drugs are unable to reach the CNS in therapeutically relevant concentration, making the BBB one of the major impediments in the treatment of CNS disorders. The significant scientific and industrial interest in the physiology and pathology of the BBB led to the development of several in vitro models of the BBB. These models are mainly based on the culture of cerebral endothelial cells. The best in vitro models which mimic the best way the in vivo anatomical conditions are the co-culture models in which brain endothelial cells are co-cultured with astrocytes and/or pericytes. Our in vitro BBB model is characterized by high transendothelial electrical resistance (TEER regularity above 200 Ohm*cm2), low permeability and expression of several transporters. Our experiments have proven that the model is suitable for basic research and for testing the interaction between the BBB and potential drug candidates (toxicity, permeability, interaction with efflux transporters) as well.