Extracellular vesicles (EVs) act in cell-to-cell communication, delivering cargo from donor to recipient cells and modulating their physiological condition. EVs secreted by leukemic blasts in patients with leukemia have been shown to influence the fate of recipient cells in the bone marrow microenvironment. Methods to quantify and to characterize them phenotypically are therefore urgently needed to study their functional role in leukemia development and to evaluate their potential as targets for therapy. We have used cryo-electron microscopy to study morphology and size of leukemic EVs, and nanoparticle tracking analysis and fluorescence triggering flow cytometry to quantify EVs in platelet-free plasma from a small cohort of leukemia patients and healthy blood donors. Additional studies with a capture bead-based assay allowed us to establish phenotypic signatures of leukemic EVs from 17 AML and 3 B-ALL patients by evaluating the expression of 37 surface antigens. In addition to tetraspanins and lineage-specific markers we found several adhesion molecules (CD29, and CD146) to be highly expressed by EVs from B-ALL and several leukemic stem cell antigens (CD44, CD105, CD133, and SSEA-4) to be expressed by EVs from AML patients. Further improvements in analytical methods to study EVs are needed before potentially using them as biomarkers for leukemia prognosis and follow-up.