Alterations in endothelial permeability are a hallmark of inflammation as well as the underlying cause of many clinical syndromes. Quantifying changes in endothelial barrier properties to water and macromolecules can be an important means of assessing the degree of cellular injury and, conversely, the effect of therapies to attenuate the inflammatory cascade. We use a combination of an isolated organ system and two cell culture models to investigate mechanisms of endothelial barrier regulation under variety of experimental conditions. Each assay has its own experimental strengths and limitations and must be used appropriately for the questions being asked. When used collectively, they can provide significant insight into the molecular regulation of lung endothelial permeability.