Mass spectrometry-based proteomics enables accurate measurement of the modulations of proteins on a large scale upon perturbation and facilitates the understanding of the functional roles of proteins in biological systems. It is a particularly relevant methodology for studying Leishmania spp., Trypanosoma cruzi and Trypanosoma brucei, as the gene expression in these parasites is primarily regulated by posttranscriptional mechanisms. Large-scale proteomics studies have revealed a plethora of information regarding modulated proteins and their molecular interactions during various life processes of the protozoans, including stress adaptation, life cycle changes and interactions with the host. Important molecular processes within the parasite that regulate the activity and subcellular localisation of its proteins, including several co- and post-translational modifications, are also accurately captured by modern proteomics mass spectrometry techniques. Finally, in combination with synthetic chemistry, proteomic techniques facilitate unbiased profiling of targets and off-targets of pharmacologically active compounds in the parasites. This provides important data sets for their mechanism of action studies, thereby aiding drug development programmes.