This study aims to optimize the isolation of exosomes from cell culture supernatant and human plasma. The authors compared various exosome isolation protocols, including ultracentrifugation, ultrafiltration, density gradient purification, and size exclusion chromatography (SEC). They found that repeated ultracentrifugation steps can reduce the quality and yield of exosome preparations. Ultrafiltration, particularly using centrifuge-based concentrating methods, was shown to be more effective in increasing exosome yield compared to traditional ultracentrifugation. The choice of concentrating device significantly impacted the yield of isolated exosomes, with centrifuge-based methods outperforming pressure-driven devices. The study also demonstrated that SEC is comparable to density gradient purification in terms of exosome purity and can be used to isolate exosomes from complex biological fluids like plasma. The authors conclude that their optimized protocol, which combines ultrafiltration and SEC, provides a high yield of pure exosomes suitable for further analysis and research.This study aims to optimize the isolation of exosomes from cell culture supernatant and human plasma. The authors compared various exosome isolation protocols, including ultracentrifugation, ultrafiltration, density gradient purification, and size exclusion chromatography (SEC). They found that repeated ultracentrifugation steps can reduce the quality and yield of exosome preparations. Ultrafiltration, particularly using centrifuge-based concentrating methods, was shown to be more effective in increasing exosome yield compared to traditional ultracentrifugation. The choice of concentrating device significantly impacted the yield of isolated exosomes, with centrifuge-based methods outperforming pressure-driven devices. The study also demonstrated that SEC is comparable to density gradient purification in terms of exosome purity and can be used to isolate exosomes from complex biological fluids like plasma. The authors conclude that their optimized protocol, which combines ultrafiltration and SEC, provides a high yield of pure exosomes suitable for further analysis and research.