The physiological roles of the granzymes A and K have been debated, especially concerning their involvement in cytotoxic and inflammatory processes. By performing N-terminal COFRADIC assisted N-terminomics on the homologous human granzymes A and K, we here provide detailed data on their substrate repertoires, their specificities, and differences in efficiency by which they cleave their substrates, all of which may aid in elucidating their key substrates. In addition, the so far uncharacterized mouse granzyme K was profiled alongside its human orthologue. While the global primary specificity profiles of these granzymes appear quite similar as they revealed only subtle differences and pointed to substrate occupancies in the P1, P1', and P2' position as the main determinants for substrate recognition, differential analyses unveiled distinguishing substrate subsite features, some of which were confirmed by the more selective cleavage of specifically designed probes.