Leishmania spp. are digenetic parasites which cause a broad spectrum of fatal diseases in humans. These parasites, as well as the other trypanosomatid, regulate gene expression at the post-transcriptional and post-translational levels, so that a poor correlation is observed between mRNA content and translated proteins. The completion of the genomic sequencing of several Leishmania species has enormous relevance to the study of the leishmaniasis pathogenesis. The combination of the available genomic resources of these parasites with powerful high-throughput proteomic analysis has shed light on various aspects of Leishmania biology as well as on the mechanisms underlying the disease. Diverse proteomic approaches have been used to describe and catalogue global protein profiles of Leishmania spp., reveal changes in protein expression during development, determine the subcellular localization of gene products, evaluate host-parasite interactions and elucidate drug resistance mechanisms. The characterization of these proteins has advanced, although many fundamental questions remain unanswered. Here, we present a historic review summarizing the different proteomic technologies applied to the study of Leishmania parasites during the last decades and we discuss the proteomic discoveries that have contributed to the understanding of Leishmania parasites biology and leishmaniasis.