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.