Studies on drug entry into the brain and permeation of the blood-brain barrier start to gain more and more importance in neuropharmaceutical research in order to develop new drugs for the therapy of central nervous system diseases. Procedures that provide quick access to permeation properties of those drugs with high throughput are difficult to achieve with animal models. Although various useful cell culture models approaching this issue have been described, results are often not comparable among each other unless determined with an equal experimental setup. Reproducibility of cell culture methods as well as corresponding findings gathered with these tools are often impeded due to the lack of details in experimental manuals. Here we present a precise manual for preparation and maintenance of porcine brain microvessel endothelial cells, serving as a culture model of the blood-brain barrier. Furthermore experimental details for blood-brain barrier transport investigations are presented. Validation of this model was carried out by determination of bioelectric properties and permeation experiments using various marker molecules reflecting paracellular and transcellular blood-brain barrier penetration. Results obtained with our model are closely resembling the in vivo-situation although astrocytes are not included. This simplification of the system is one of the major advantages towards robot derived cell cultures necessary for high throughput screening.