Embodiments of the present invention provide an in vitro assay that measures cell monolayer permeability. The assay utilizes detection of a binding pair initially separated by the cell monolayer as a qualitative and quantitative measure of the permeability of the monolayer. In certain embodiments, the assay is performed in the presence of a test substance that may or may not affect cell permeability. In particular aspects, the assay is performed on a substrate that is elastic. One can also assay spatial resolution of local changes by visualizing regional variations of the cell monolayers.