Proteases play a crucial role in processing or breaking down proteins. They exhibit varying levels of substrate specificity that determine their physiological or pharmacological functions. However, this specificity is not well-understood. To address this, we have developed hiMAPS (high-throughput mapping of protease cleavage sites), a mass spectrometry-based method that can survey tens of thousands of distinct peptides. By applying this approach to human DPP4 and C. elegans DPF-3, two prolyl peptidases from the Dipeptidyl Peptidase IV (DPPIV) family, we have derived quantitative models that confirm a strong preference for Pro and Ala in the penultimate (P1) position from the N-terminus, but also reveal differences in the relative preferences for other residues. The hiMAPS approach can be applied to other exoproteases using various cheap sources of highly diverse peptides. Additionally, we have observed that these proteases tolerate and accept substrates containing Thr and Ser at P1 position.