Lakewater microorganisms secrete proteases which contribute to the turnover of dissolved organic matter and the degradation of peptidic contaminants. However, little is known about the identities and substrate specificities of these proteases. Herein, we sought to characterize the global proteolytic fingerprint of the extracellular proteases present in Lake Geneva, the largest freshwater body in Central Europe. Using Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS), we identified preferred enzymatic cleavage next to positively charged and certain nonpolar amino acids, while cleavage next to negatively charged residues was disfavored. Specifically, the detected dominant cleavage sites were surrounded by arginine and lysine, consistent with a trypsin-like substrate specificity. This pattern was conserved across seasons and water depths and was shared with two other Swiss lakes. In contrast, we observed variability in the numbers and types of less prevalent cleavage sites across samples, suggesting that the degree of heterogeneity in proteolytic substrate specificity varies spatially and temporally. Using class-specific inhibitors, we found that serine and metalloproteases contribute to both exo-and endoproteolytic activity in lakewater. Our findings expand our understanding of protein stability in lake ecosystems and may be used to predict the fate of peptidic contaminants in the environment.