This chapter provides a comprehensive guide on the isolation and characterization of exosomes from cell culture supernatants and biological fluids. Exosomes are small vesicles secreted by various cell types, formed through inward budding of endocytic compartments and released into the extracellular medium. Their physiological functions include eliminating proteins no longer needed in differentiated cells and facilitating intercellular communication, such as antigen transfer between antigen-bearing cells and antigen-presenting cells. The chapter outlines several protocols for purifying exosomes, including differential ultracentrifugation, filtration, and immunoisolation. Each protocol details the steps for collecting and processing exosome-containing supernatants, ensuring the purity and quality of the isolated vesicles. Key steps include centrifugation at high speeds to pellet exosomes, washing to remove contaminants, and additional purification steps for enhanced purity. Characterization methods, such as electron microscopy, SDS-PAGE, immunoblotting, and FACS analysis, are described to assess the quality and composition of exosome preparations. These methods help confirm the presence of exosomal proteins and ensure that the isolated vesicles are indeed exosomes rather than other contaminants. The chapter also provides detailed protocols for preparing exosome production medium to avoid contamination from serum-derived exosomes and for purifying exosomes from viscous fluids like plasma. Additionally, it covers techniques for immunolabeling exosomes for electron microscopy and determining the density of exosome preparations on sucrose gradients. Overall, the chapter serves as a practical resource for researchers aiming to isolate and characterize exosomes for various applications, including functional studies and clinical research.This chapter provides a comprehensive guide on the isolation and characterization of exosomes from cell culture supernatants and biological fluids. Exosomes are small vesicles secreted by various cell types, formed through inward budding of endocytic compartments and released into the extracellular medium. Their physiological functions include eliminating proteins no longer needed in differentiated cells and facilitating intercellular communication, such as antigen transfer between antigen-bearing cells and antigen-presenting cells. The chapter outlines several protocols for purifying exosomes, including differential ultracentrifugation, filtration, and immunoisolation. Each protocol details the steps for collecting and processing exosome-containing supernatants, ensuring the purity and quality of the isolated vesicles. Key steps include centrifugation at high speeds to pellet exosomes, washing to remove contaminants, and additional purification steps for enhanced purity. Characterization methods, such as electron microscopy, SDS-PAGE, immunoblotting, and FACS analysis, are described to assess the quality and composition of exosome preparations. These methods help confirm the presence of exosomal proteins and ensure that the isolated vesicles are indeed exosomes rather than other contaminants. The chapter also provides detailed protocols for preparing exosome production medium to avoid contamination from serum-derived exosomes and for purifying exosomes from viscous fluids like plasma. Additionally, it covers techniques for immunolabeling exosomes for electron microscopy and determining the density of exosome preparations on sucrose gradients. Overall, the chapter serves as a practical resource for researchers aiming to isolate and characterize exosomes for various applications, including functional studies and clinical research.