Endothelial cells lining the vessels of the vasculature and the cell–cell junctions, which join them, �provide the primary barrier to the passage of fluids, immune cells, and macromolecules between the bloodstream and the tissues. Appropriate and dynamic regulation of this barrier is required during normal �physiological processes; however, if not tightly controlled, increased permeability of the endothelium can also contribute to many pathological situations, including chronic inflammatory diseases and edema. The development of in vitro methods to study endothelial barrier function has been key in the identification of molecular mechanisms underlying many of these disease states. In this chapter, we describe three complementary approaches to measure endothelial monolayer permeability and barrier function in vitro.