Disclosed are methods and compositions for detecting pregnancy in an animal by means of assaying peptidase activity of one or more Pregnancy Associated Glycoproteins (PAGs). In certain aspects, methods also comprising use of an antibody that binds immunologically to a PAG that displays proteolytic activity are provided. Substrates of proteolytic PAGs are also provided, as are kits, and methods of use. Further, methods of purifying PAGs based on their proteolytic activity are also provided.