Endothelial cells line the inner surface of all blood vessels and constitute a selective barrier between blood and tissue. Permeation of solutes across the endothelial cell monolayer occurs either paracellularly through specialized endothelial cell-cell junctions or transcellularly via special transport mechanisms including transcytosis, via the formation of transcellular channels, or by cell membrane transport proteins. Several in vitro assays have been developed in the past few decades to analyze the molecular mechanisms of transendothelial permeability. Measurement of the electrical resistance of the cell monolayer has proven to be particularly suitable for analyzing paracellular barrier function with high-time resolution over long time periods. We review the various permeability assays and focus on the electrical impedance analysis of endothelial cell monolayers. We also address current progress in the development of techniques used to investigate endothelial permeability with high-lateral resolution and under mechanical loads.