Using a combination of mass spectrometry-based quantitative proteomics, chemical and metabolic labelling, we provide the first protein-level evidence that the Hsp90 inhibition affects the expression of hundreds of proteins in Leishmania spp. Our study defines the Leishmania parasite’s response to Hsp90 inhibition at its global proteome and provides a rich resource for future studies on Leishmania biology and antileishmanial drug development.