Immunoconjugates targeting cell-surface antigens have demonstrated clinical activity to enable regulatory approval in several solid and hematologic malignancies. We hypothesize that a rigorous and comprehensive surfaceome profiling approach to identify osteosarcoma-specific cell-surface antigens can similarly enable development of effective therapeutics in this disease. Herein, we describe an integrated proteomic and transcriptomic surfaceome profiling approach to identify cell-surface proteins that are highly expressed in osteosarcoma but minimally expressed on normal tissues. Using this approach, we identified targets that are highly expressed in osteosarcoma. Three targets, MT1-MMP, CD276, and MRC2, were validated as overexpressed in osteosarcoma. Further, we tested BT1769, an MT1-MMP-targeted Bicycle toxin conjugate, in osteosarcoma PDX models. The results showed BT1769 had encouraging anti-tumor activity, high affinity for its target and a favorable pharmacokinetic profile. This confirms the hypothesis that our approach identifies novel targets with significant therapeutic potential in osteosarcoma.