This review article provides a comprehensive overview of the classical and state-of-the-art methods for isolating extracellular vesicles (EVs), including exosomes. EVs play a crucial role in cell communication and the transport of diagnostic molecules, making their isolation and analysis essential for various biological and medical applications. The review highlights the advantages and disadvantages of different isolation methods, such as ultracentrifugation, density gradient ultracentrifugation, filtration techniques (ultrafiltration, hydrostatic dialysis, gel filtration), and methods based on changes in EV solubility and aggregation (precipitation with polyethylene glycol, protamine, sodium acetate, and organic solvents). Each method is evaluated based on isolation efficiency, yield, purity, and the presence of contaminants. The article emphasizes the importance of selecting the appropriate method based on the specific requirements of the experiment, such as the need for high yield, purity, or specific markers. The review also discusses the limitations and challenges of current isolation methods, such as the difficulty in achieving pure EV fractions and the need for further validation of new techniques. Overall, the article underscores the ongoing efforts to develop more efficient and standardized methods for EV isolation to advance the field of biomarker discovery and disease diagnosis and therapy.This review article provides a comprehensive overview of the classical and state-of-the-art methods for isolating extracellular vesicles (EVs), including exosomes. EVs play a crucial role in cell communication and the transport of diagnostic molecules, making their isolation and analysis essential for various biological and medical applications. The review highlights the advantages and disadvantages of different isolation methods, such as ultracentrifugation, density gradient ultracentrifugation, filtration techniques (ultrafiltration, hydrostatic dialysis, gel filtration), and methods based on changes in EV solubility and aggregation (precipitation with polyethylene glycol, protamine, sodium acetate, and organic solvents). Each method is evaluated based on isolation efficiency, yield, purity, and the presence of contaminants. The article emphasizes the importance of selecting the appropriate method based on the specific requirements of the experiment, such as the need for high yield, purity, or specific markers. The review also discusses the limitations and challenges of current isolation methods, such as the difficulty in achieving pure EV fractions and the need for further validation of new techniques. Overall, the article underscores the ongoing efforts to develop more efficient and standardized methods for EV isolation to advance the field of biomarker discovery and disease diagnosis and therapy.