CDCP1 is an oncogenic orphan transmembrane receptor and a promising target for detection and treatment of cancer. Extracellular proteolysis of CDCP1 by poorly defined mechanisms induces pro-metastatic signaling. We describe a novel approach for rapid identification of proteases responsible for key proteolytic events exploiting a substrate-biased activity-based probe (sbABP) that incorporates a substrate cleavage motif grafted onto a peptidyl-diphenyl-phosphonate warhead for specific target protease capture, isolation and identification. Using a CDCP1-biased probe we identify urokinase (uPA) as the master regulator of CDCP1 proteolysis, both by direct cleavage and via activation of CDCP1-cleaving plasmin. We show that co-expression of uPA and CDCP1 is strongly predictive of poor disease outcome across multiple cancers and demonstrate that uPA-mediated CDCP1 proteolysis promotes metastasis in disease-relevant preclinical in vivo models. These results highlight CDCP1 cleavage as a potential target to disrupt cancer and establish sbABP technology as a new approach to identify disease-relevant proteases.