The metalloproteinase ovastacin is released by the mammalian oocyte upon fertilization and cleaves zona pellucida protein 2, a component of the surrounding extracellular matrix, at a distinct cleavage site. This limited proteolysis hardens the zona pellucida, abolishes sperm binding and thereby regulates fertility. Therefore, this process has to be tightly controlled by the plasma protein fetuin-B, an endogenous competitive inhibitor. At present, little is known about how the cleavage characteristics of ovastacin differ from closely related proteases. Physiological implications of ovastacin beyond ZP2 cleavage are still obscure. In this study, we employed N-terminal amine isotopic labeling of substrates (N-TAILS) contained in the secretome of mouse embryonic fibroblasts to elucidate the substrate specificity and the precise cleavage site specificity. Furthermore, we were able to unravel the physicochemical properties governing enzyme-substrate interactions. Eventually, we identified several potential physiological substrates with significance for mammalian fertilization. These results suggest that ovastacin might affect sperm interaction beyond zona pellucida hardening