Leishmaniasis is a life-threatening neglected tropical disease caused by over 20 species ofLeishmaniaparasites. Visceral leishmaniasis, also known as kala-azar, is particularly lethal, with a 95% mortality rate if left untreated. Currently, no vaccine is available, and chemotherapy remains the primary treatment option. However, these drugs have drawbacks such as high toxicities, the emergence of resistant strains, and high costs. Therefore, there is a need to develop new and safe treatments. Glycosomes are essential organelles for the survival ofLeishmaniaparasites. They are maintained by peroxin (PEX) proteins, which are responsible for glycosome biogenesis, including targeting proteins to glycosomes. Previous studies have shown that blocking the interaction between the import receptor PEX19 and the docking factor PEX3 killsTrypanosoma bruceiby disrupting glycosome biogenesis. In this study, we screened an FDA-approved drug repurposing library using an AlphaScreen based assay and identified inhibitors ofLdPEX3-LdPEX19 interactionin vitro. The inhibitor effectively killsLeishmaniaparasites, including the challenging amastigote forms contained within the infected mammalian host cells. This study validates the inhibition of glycosome biogenesis inLeishmaniaas a potential approach for developing new anti-leishmanial therapies.