Updated project metadata. Extracellular Vesicles (EVs) exhibit great potential in for brain disease diagnositics and treatment, representing a valuable tool in the new era of pPrecision medicine which demands high-quality human biospecimens. However, current brain exosome EV isolation approaches rely on its tissue dissociation, which may can contaminate the exosome EV fractions with immature intralumenalcellular vesicles that wcould otherwise be targeted for endolysosomal degradation. Hereby, we present an efficient exosome purification method that captures a more physiologically relevant EV population exosome-enriched extracellular vesicles (EVs) spontaneously released by mouse and human brain tissue, representing a more physiologically relevant exosome EV population.