Abstract: The present invention provides compositions and methods for preparation of a three-dimensional transendothelial cell migration (TEM) assay. These compositions and methods are uniquely suited for the high throughput TEM assay, and for the analysis and identification of TEM mediators which inhibit or stimulate this process. The composition for detecting migration of cells comprises a solid layer comprising collagen gel; a first cellular layer in contact with the solid layer and comprising a first cell type; and a second cell type seeded on top of the first cellular layer. Optionally, gelatin is included in the collagen gel. A 96 well plate format is disclosed, the combination with a high throughput cellular scanner enables high throughput TEM assay.