The increasing interest in extracellular vesicles (EVs) research is fueled by reports indicating their unique role in intercellular communication and potential connection to the development of common human diseases. The unique role assumes unique protein and nucleic acid cargo. Unfortunately, accurate analysis of EVs cargo faces a challenge of EVs isolation. Generally used isolation techniques do not separate different subtypes of EVs and even more, poorly separate EVs from non-EVs contaminants. Further development of EVs isolation protocols urgently needs a quantitative method of EVs purity assessment. We report here that multiple reaction monitoring assay using internal standards carrying peptides for quantification of EVs and non-EVs proteins is a suitable approach to assess purity of EVs preparations. As a first step in potential standardization of EVs isolation, we have evaluated polymer-based precipitation techniques and compared them to traditional ultracentrifugation protocol.