There is significant interest in engineering proteases with desired proteolytic properties. We describe a high-throughput fluorescence-activated cell sorting (FACS) assay for detecting altered proteolytic activity of protease in yeast， at the single cell level. This assay relies on coupling yeast endoplasmic reticulum (ER) retention， yeast surface display， and FACS analysis. The method described here allows facile screening of large libraries， and of either protease or substrate variants， including the screening of protease libraries against substrate libraries. We demonstrate the application of this technique in the screening of libraries of Tobacco Etch Virus protease (TEV-P) for altered proteolytic activities. In addition， the generality of this method is also validated by other proteases such as human granzyme K and the hepatitis C virus protease， and the human Abelson tyrosine kinase.