Extracellular vesicles (EV) gained considerable interest in recent years as both diagnostic tools and templates for therapeutic applications. EVs carry a number of cell-specific markers which gave researchers the opportunity of employing them as liquid biopsies causing no discomfort to patients. On the other hand, they are very exciting candidates for drug delivery due to their eobiotic origin, physicochemical and size characteristics. Isolation of EVs is performed by several strategies, having advantages and disatvantages over each other. As such, the method of EV isolation and in particular exosome isolation determines the quality and purity of obtained vesicles. In this chapter, extracellular vesicle isolation methods are evaluated with regard to their further use. Methods such as ultracentrifugation with different modifications, size exclusion chromatography, ultrafiltration, affinity and precipitation are compared with respect to the yield efficacy and purity of isolates. Furthermore, the advantages and disadvantages of different methods according to the purpose of use are revealed. Recent progress and remaining challenges in the isolation of EVs with regard to diagnosis and treatment is reviewed and discussed. In order to select the most suitable method researchers should clearly define purity, yield, quantity and quality requirements for exosomes, and consider disadvantages of distinct isolation methods.