The blood–nerve barrier (BNB) is one of the functional barriers sheltering the nervous system from circulating blood. It is very important to understand the cellular properties of endothelial cells of endoneurial origin because these cells constitute the bulk of the BNB. This chapter describes a standard protocol for isolating the endothelial cells forming the BNB. In addition, methods for confirming some of the barrier properties of isolated endothelial cells are also described.