Mapping the phospho-catalytic profile of kinase enzymes in cells or tissues remains a challenge. Here, we introduce a practical biochemical assay to measure the enzymatic activity of kinases using peptides as surrogate sensors to identify kinases in tumor biopsies and cell lines. The platform relies on collections of peptide probes that are derived from biological target sites of kinases, and that operate as distinct combinatorial peptide sets to simultaneously distinguish and measure the phospho-catalytic activity of many kinase enzymes. The assay is modular by design: users can adapt probe libraries and assay conditions to their needs. We named this functional proteomic platform 'High-Throughput Kinase Activity Mapping (HT-KAM) system'. The procedure described in this Protocol Exchange chapter focuses on detailing the biochemical assay, and is related to the Nature Cell Biology manuscript NCB-C36710 titled: "Mapping phospho-catalytic dependencies of therapy-resistant tumors reveals actionable vulnerabilities".