Updated project metadata. Extracellular vesicles (EVs) are lipid bilayered vesicles released into extracellular milieu by most of cells. Currently, various purification methods of EVs have been established but most of studies use ultracentrifugation-based purification of EVs. However, these methods have the problem of co-purification with non-vesicular contaminants including various different types of particles, small apoptotic vesicles, protein aggregates, and other derived from dead cells or abundant extracellular proteins. Therefore, evaluation of non-vesicular contaminants gives valuable information in EV research. Here, we purified EVs with high purity using density gradient ultracentrifugation and then purified EVs were treated by trypsin to discriminate extra-vesicular proteins from intra-vesicular proteins.