Background: Low survival rates in metastatic high-grade osteosarcoma (HGOS) have remained stagnant for the last three decades. This study aims to investigate the role of aminopeptidase N (ANPEP) in HGOS progression and its targeting with a novel lipophilic peptidase-enhanced cytotoxic compound melphalan flufenamide (melflufen) in HGOS. Methods: Meta-analysis of publicly available gene expression datasets was performed to determine the impact of ANPEP gene expression on metastasis-free survival of HGOS patients. The efficacy of standard-of-care anti-neoplastic drugs and a lipophilic peptidase-enhanced cytotoxic conjugate melflufen was investigated in patient-derived HGOS ex vivo models and cell lines. The kinetics of apoptosis and necrosis induced by melflufen and doxorubicin were compared. Anti-neoplastic effects of melflufen were investigated in vivo. Results: Elevated ANPEP expression in diagnostic biopsies of HGOS patients was found to significantly reduce metastasis-free survival. In drug sensitivity assays, melflufen has shown an anti-proliferative effect in HGOS ex vivo samples and cell lines, including those resistant to methotrexate, etoposide, doxorubicin, and PARP inhibitors. Further, HGOS cells treated with melflufen displayed a rapid induction of apoptosis and this sensitivity correlated with high expression of ANPEP. In combination treatments, melflufen demonstrated synergy with doxorubicin in killing HGOS cells. Finally, Melflufen displayed anti-tumor growth and anti-metastatic effects in vivo. Conclusion: This study may pave the way for use of melflufen as an adjuvant to doxorubicin in improving the therapeutic efficacy for the treatment of metastatic HGOS.