Exosomes are small endosome-derived lipid nanoparticles (50-120 nm in diameter), actively secreted by exocytosis in most living cells. Recently, there is a growing interest of research focused on studying the exosome functions and to understand ways to use them for therapeutic applications in a wide variety of disorders, such as cancer, cardiovascular, neurodegenerative, and musculoskeletal diseases. Recently, a number of techniques have been developed for the isolation of exosomes such as ultracentrifugation, micro-filtration centrifugation, gradient centrifugation, and size-exclusion chromatography. In this chapter, we reveal the protocol and key insights into the isolation, purification, and characterization of exosomes using ultracentrifugation method.