Despite their clinical relevance and role in parasitic virulence, exosomes produced by the protozoan parasite Leishmania lack specific proteomic biomarkers. Herein, we utilized utilized serial differential ultracentrifugation to separate three distinct fractions of Leishmania-derived extracellular vesicles and validated their properties using nanoparticle tracking analysis and transmission electron microscopy. Proteomic analysis was then used to assess their protein contentand identify novel biomarkers.