As proteases sculpt the proteome in both homeostatic and pathogenic processes, unraveling their primary signaling pathways and key substrates is of utmost importance. Hence, with the development of procedures enriching for proteolysis-indicative peptides and the availability of more sensitive mass spectrometers, protease degradomics technologies are ideally suited to gain insight into a protease's substrate repertoire and substrate-specificity profile. Especially, knowledge on discriminating sequence features among closely related homologues and orthologues may aid in identifying key targets and developing protease-specific inhibitors. Although clever labeling strategies allow one to compare the substrate repertoires and critical protease substrate recognition motifs of several proteases in a single analysis, comprehensive views of (differences in) substrate subsite occupancies of entire protease families is lacking. Therefore, we here describe a hierarchical cluster analysis of the positional proteomics determined cleavage sites of a family of serine proteases: the granzymes. We and others previously assigned clear murine orthologues for all 5 human granzymes. As such, hierarchical clustering of the sequences surrounding granzyrne cleavage sites reveals detailed insight into granzyme-specific differences in substrate selection and thereby deorphanizes the substrate specificity profiles and repertoires of the human and murine orthologous granzymes A, B, H/C, M, and K. KEYWORDS: granzyme, positional proteomics, degradomics, N-terminal COFRADIC, protease, substrate, substrate specificity profiles and repertoires of the human and murine orthologous granzymes A, B, H/C, M, and K.