Abstract Next-generation Trop-2-targeted therapy against advanced cancers is hampered by expression of Trop-2 in normal tissues. We discovered that Trop-2 undergoes proteolytic activation by ADAM10 in cancer cells, leading to the exposure of a previously inaccessible protein groove flanked by two N-glycosylation sites. We designed a recognition strategy for this region, to drive selective cancer vulnerability in patients. Most undiscriminating anti-Trop-2 monoclonal antibodies (mAb) recognize a single immunodominant epitope. Hence, we removed it by deletion-mutagenesis. Cancer-specific, glycosylation-prone mAb were selected by ELISA, bio-layer interferometry, flow cytometry, confocal microscopy for differential binding to cleaved/activated, wild-type and glycosylation-site-mutagenized Trop-2. The resulting 2G10 mAb family binds Trop-2-expressing cancer cells, but not Trop-2 on normal cells. We humanized 2G10 by state-of-the-art CDR grafting/re-modeling, yielding Hu2G10. This antibody binds cancer-specific, cleaved/activated Trop-2 with Kd <10-12 M, and uncleaved/wtTrop-2 in normal cells with Kd 3.16x10-8 M, thus promising an unprecedented therapeutic index in patients. In vivo, Hu2G10 ablates growth of Trop-2-expressing breast, colon, prostate cancers, but shows no evidence of systemic toxicity, paving the way for a paradigm shift in Trop-2-targeted therapy.