Exosomes derived from mesenchymal stem cells regulate cell migration, proliferation, differentiation, and synthesis of the extracellular matrix. The ultracentrifugation method is the gold standard for exosome isolation with its simple protocol, high exosomal yield, allowing to work with high volume of sample features, but low purity and requirement of specialized equipment. Optimal isolation does not exist yet for obtaining the exosomes derived from mesenchymal stem cells for clinical usage. We hypothesized that magnetic-activated cell sorting may provide, effective and rapid exosomes derived from human mesenchymal stem cells when compared to ultracentrifugation. We, therefore, aimed to compare the efficiency of magnetic-activated cell sorting and ultracentrifugation for human bone marrow mesenchymal stem cell-derived exosome isolation from culture media by protein quantification, surface biomarker analysis, particle size and number analysis, and morphological analysis in vitro. Magnetically activated cell sorting provided a higher purity and amount of exosome when compared to ultracentrifugation. Magnetically activated cell sorting isolated samples exhibited the presence of magnetic beads morphologically. The particle number of the magnetic-activated cell sorting group was higher than the ultracentrifugation. In conclusion, the isolation of exosomes derived from mesenchymal stem cells by magnetic-activated cell sorting presents a quick and reliable method to collect them at high purity for clinical usage.