Significance Cell surface proteolysis is a key mechanism for cell-cell communication and cellular signaling that is commonly dysregulated in human disease. Proteolytic cleavage events at the cell surface often evade detection by conventional proteomics methods owing to their low relative abundance compared to cytoskeletal and cytoplasmic proteins. Here we address this limitation by developing a new enzymatic tool, subtiligase-TM, for targeted mapping of cell surface proteolysis. We combine subtiligase-TM with quantitative proteomics to map proteolytic cleavage sites on the cell surface with single amino acid resolution. Based on the importance of cell surface proteolysis in human health and disease, subtiligase-TM opens up new opportunities for identifying biomarkers and therapeutic targets.