Mesenchymal stromal cells (MSCs) derived EVs are greatly studied in disease models but lack of manifested characterization rather than generic EVs characterization in different EV subpopulations, greatly limits our ability to bring the most reliable EV types into our studies. We provide a comparative evaluation between three most well-reputed EVs subpopulations isolated by ultracentrifuge (EV-110K), sucrose gradient (EV-S110K), and 20,000g centrifugation only (EV-20K). We delved into purity, membrane perfectness, genuine content and apoptotic markers as prior factors need to be checked before functional application. Based on our observations, EV-SUC confirmed higher membrane integrity compare to EV-20K (P<0.01) and EV-110K (P<0.05) and carried the parental content like as acetylcholinesterase (AChE), however it requires to be adjusted in yield of purity. Based on these findings, EV-S110K as selected EV, also showed promising functional results on healthy and diabetic islet derived cell. Proteome profiling also confirmed distinct protein content of these three sub-populations. Our results attempt to stand up for the necessity of determining the most practical EV subpopulations prior to check its functional tests in in vitro models or even in disease models.