Abstract <p>Intercellular communication is a critical component of maintaining tissue homeostasis. One of the communication methods is the transfer of a wide range of biologically active molecules—proteins, nucleic acids (primarily noncoding RNA) and lipids—in extracellular vesicles (EV). This allows EV-producing cells to change the metabolic and transcriptional activity of individual target cell populations over a wide range and “tune” it in accordance with tissue needs. Most of the existing studies are focused on the composition and functions of EVs secreted into the external environment, while the study of the membrane-associated vesicle subclass (membrane-associated vesicles, MAV) is hampered by the lack of generally accepted methods for their isolation from different cell types, including cultured ones, and characterization. In this paper, we present a protocol for isolating the MAV fraction of cultured mesenchymal stromal/stem cells while preserving the morphology and viability of the cells themselves using hyaluronidase treatment. This protocol demonstrated the highest efficiency compared to other tested methods and allows one to obtain a fraction significantly enriched in vesicles, as demonstrated by nanoparticle tracking analysis and transmission electron microscopy. A comparative analysis of the characteristics of MAV and EV secreted into the medium showed that the proposed MAV isolation protocol allows one to obtain a fraction with a similar particle concentration, but they are smaller in size compared to EV. Thus, the isolation method we described allows one to obtain a MAV fraction for further analysis of their composition and functional features.</p>