Exosomes are membrane-bound vesicles secreted by eukaryotic cells, formed within multivesicular bodies (MVBs) and released when these compartments fuse with the plasma membrane. They play a crucial role in intercellular communication by transferring proteins, lipids, and nucleic acids. The biogenesis of exosomes involves complex molecular mechanisms, including the ESCRT machinery, lipids, and tetraspanins, which facilitate the formation of intraluminal vesicles (ILVs) within MVBs. ESCRT proteins, such as TSG101 and ALIX, are essential for ILV formation and exosome secretion. However, ESCRT-independent mechanisms, involving lipids like ceramide and phosphatidic acid, and tetraspanins, also contribute to exosome biogenesis. Exosome secretion requires the transport and fusion of MVBs with the plasma membrane, a process regulated by RAB proteins, such as RAB27A, which is crucial for exosome release. SNARE proteins are involved in the fusion of MVBs with the plasma membrane. Different types of exosomes, such as those derived from MVBs and those from the plasma membrane, have distinct compositions and functions. The heterogeneity of exosomes and their subtypes highlights the need for improved methods to separate and characterize them. Current protocols for exosome purification often co-purify different EV subtypes, necessitating the development of more precise techniques. Understanding the molecular mechanisms of exosome biogenesis and secretion is essential for targeting their production and function in various biological contexts.Exosomes are membrane-bound vesicles secreted by eukaryotic cells, formed within multivesicular bodies (MVBs) and released when these compartments fuse with the plasma membrane. They play a crucial role in intercellular communication by transferring proteins, lipids, and nucleic acids. The biogenesis of exosomes involves complex molecular mechanisms, including the ESCRT machinery, lipids, and tetraspanins, which facilitate the formation of intraluminal vesicles (ILVs) within MVBs. ESCRT proteins, such as TSG101 and ALIX, are essential for ILV formation and exosome secretion. However, ESCRT-independent mechanisms, involving lipids like ceramide and phosphatidic acid, and tetraspanins, also contribute to exosome biogenesis. Exosome secretion requires the transport and fusion of MVBs with the plasma membrane, a process regulated by RAB proteins, such as RAB27A, which is crucial for exosome release. SNARE proteins are involved in the fusion of MVBs with the plasma membrane. Different types of exosomes, such as those derived from MVBs and those from the plasma membrane, have distinct compositions and functions. The heterogeneity of exosomes and their subtypes highlights the need for improved methods to separate and characterize them. Current protocols for exosome purification often co-purify different EV subtypes, necessitating the development of more precise techniques. Understanding the molecular mechanisms of exosome biogenesis and secretion is essential for targeting their production and function in various biological contexts.