Extracellular vesicles (EVs) are important mediators of cell signaling pathways that contain diverse biological information such as DNAs, RNAs, lipids, and proteins. It is attracting attention in the diagnosis, treatment, basic research, and clinical applications of various diseases. Therefore, accurate and robust quantitation methods for size, concentration, and other properties such as protein quantification are essential. However, current analytical techniques have limitations in providing sufficient information on EVs in a single cell level. In this work, we used mass cytometry to develop analytical protocol of exosomes analysis in a single cell level, via immunostaining using CD63, a biomarker of exosome, to phenotype exosomes. Measurement of single exosomes is important as it provides information about the heterogeneity and subgroups of exosomes. However, there are limitations in detecting exosomes with very small size and low refractive index. To overcome this problem, quantum dot (Qdot)-conjugated antibody was also introduced instead of a traditional fluorescence antibody or metal conjugated antibody. As a crosschecking approach, the size distribution and number concentration of exosomes derived from A549 cells were measured using the conventional nanoparticle tracking analysis (NTA) methods and then compared with the results of mass cytometry, such as the expression and phenotype of exosomes in various subtypes.