Several mechanisms of cancer progression, from tumour onset to metastasis, critically involve proteolytic activity. Studying the role of proteases in cancer, it is important to consider single nucleotide variants (SNVs) affecting the active site of proteases, that might impact cleavage specificity, substrate processing and thus cancer cell behaviour. To facilitate systematic studies, we here present a targeted approach to determine the impact of cancer-associated protease variants (TACAP). Starting with the semi-automated identification of potential specificity-modulating SNVs, our workflow comprises mass spectrometry-based cleavage specificity profiling and substrate identification, localisation and inhibitor studies, followed by functional analyses investigating cancer cell migration. We demonstrate the feasibility of TACAP by analysing the meprin β R238Q variant. The amino acid exchange R238Q leads to a loss of meprin β’s characteristic cleavage preference for acidic amino acids at the P1’ position, accompanied with changes in substrate pool and inhibitor affinity compared to meprin β wildtype.