Abstract: Presented herein are polypeptide substrates based on Notch polypeptides, assay methods based on the use of these substrates, and screening methods directed toward identifying inhibitors of ?-secretase activity. The assay methods and the screening methods are adapted for use in high throughput multi-well plate assay apparatuses. In many embodiments the substrate polypeptides are labeled for ease of detection, and/or may bind specific ligands that themselves are labeled. Generally the labels promote high specificity as well as high sensitivity of detection. These features render the assay and screening methods that employ the labeled substrates especially suited for use in high throughput assay formats.