Extracellular vesicles (EVs) are membrane-bound nanoparticles involved in cell communication and transport of diagnostic molecules. Various methods exist for EV isolation, including ultracentrifugation, filtration, and precipitation with polymers or organic solvents. Each method has advantages and disadvantages in terms of efficiency, purity, and applicability. Ultracentrifugation is a classical method that separates EVs by buoyant density but is time-consuming and requires specialized equipment. Density gradient ultracentrifugation improves separation but is complex and labor-intensive. Filtration methods, such as ultrafiltration and hydrostatic dialysis, are faster and less expensive but may result in lower purity. Precipitation with polyethylene glycol (PEG) or protamine is efficient and simple but can lead to contamination with non-EV proteins. The PROSPR method, which uses organic solvent to precipitate proteins, isolates EVs with high purity but may cause membrane fusion. Distributive methods using PEG and dextran aim to reduce protein contamination by preferentially accumulating EVs in one phase. Despite advancements, no universal EV isolation method exists, and the choice of method depends on the specific application, such as diagnostic or therapeutic use. EVs are valuable for disease biomarkers and drug delivery, but their isolation remains a challenge due to heterogeneity and contamination issues.Extracellular vesicles (EVs) are membrane-bound nanoparticles involved in cell communication and transport of diagnostic molecules. Various methods exist for EV isolation, including ultracentrifugation, filtration, and precipitation with polymers or organic solvents. Each method has advantages and disadvantages in terms of efficiency, purity, and applicability. Ultracentrifugation is a classical method that separates EVs by buoyant density but is time-consuming and requires specialized equipment. Density gradient ultracentrifugation improves separation but is complex and labor-intensive. Filtration methods, such as ultrafiltration and hydrostatic dialysis, are faster and less expensive but may result in lower purity. Precipitation with polyethylene glycol (PEG) or protamine is efficient and simple but can lead to contamination with non-EV proteins. The PROSPR method, which uses organic solvent to precipitate proteins, isolates EVs with high purity but may cause membrane fusion. Distributive methods using PEG and dextran aim to reduce protein contamination by preferentially accumulating EVs in one phase. Despite advancements, no universal EV isolation method exists, and the choice of method depends on the specific application, such as diagnostic or therapeutic use. EVs are valuable for disease biomarkers and drug delivery, but their isolation remains a challenge due to heterogeneity and contamination issues.