Extracellular vesicles (EVs), including exosomes and microvesicles, are membrane-bound particles released by cells that serve as vehicles for intercellular communication by transferring proteins, lipids, and RNA between cells. Exosomes originate from multivesicular endosomes (MVEs) and are typically 40–100 nm in size, while microvesicles are larger and originate directly from the plasma membrane. Despite their importance in cell communication, the molecular mechanisms of EV formation, targeting, and function remain poorly understood, and current methods for isolating and analyzing EVs are not fully capable of distinguishing between exosomes and microvesicles. This review discusses the characterization of EVs, their formation mechanisms, and the challenges in identifying their physiological roles. It also covers the isolation and characterization techniques for EVs, including methods such as differential ultracentrifugation, immunoadsorption, and cryo-electron microscopy. The molecular composition of EVs includes proteins, lipids, and RNA, with exosomes enriched in cholesterol, sphingomyelin, and hexosylceramides. EVs can carry mRNA and miRNA, which can be translated by recipient cells, and their RNA content varies depending on the cell type and isolation method. The biogenesis of EVs involves complex molecular mechanisms, including the ESCRT machinery for exosome formation and other pathways for microvesicle formation. The release of EVs is regulated by various cellular processes, including cytoskeletal dynamics, small GTPases, and SNARE proteins. EVs interact with recipient cells through specific binding mechanisms, and their functions include antigen presentation, immune modulation, and disease progression. EVs have been implicated in various physiological and pathological processes, including cancer, neurodegenerative diseases, and infectious diseases. The study of EVs is an active area of research, with ongoing efforts to develop standardized methods for their isolation, characterization, and functional analysis. The International Society for Extracellular Vesicles was established to coordinate research and standardize the field.Extracellular vesicles (EVs), including exosomes and microvesicles, are membrane-bound particles released by cells that serve as vehicles for intercellular communication by transferring proteins, lipids, and RNA between cells. Exosomes originate from multivesicular endosomes (MVEs) and are typically 40–100 nm in size, while microvesicles are larger and originate directly from the plasma membrane. Despite their importance in cell communication, the molecular mechanisms of EV formation, targeting, and function remain poorly understood, and current methods for isolating and analyzing EVs are not fully capable of distinguishing between exosomes and microvesicles. This review discusses the characterization of EVs, their formation mechanisms, and the challenges in identifying their physiological roles. It also covers the isolation and characterization techniques for EVs, including methods such as differential ultracentrifugation, immunoadsorption, and cryo-electron microscopy. The molecular composition of EVs includes proteins, lipids, and RNA, with exosomes enriched in cholesterol, sphingomyelin, and hexosylceramides. EVs can carry mRNA and miRNA, which can be translated by recipient cells, and their RNA content varies depending on the cell type and isolation method. The biogenesis of EVs involves complex molecular mechanisms, including the ESCRT machinery for exosome formation and other pathways for microvesicle formation. The release of EVs is regulated by various cellular processes, including cytoskeletal dynamics, small GTPases, and SNARE proteins. EVs interact with recipient cells through specific binding mechanisms, and their functions include antigen presentation, immune modulation, and disease progression. EVs have been implicated in various physiological and pathological processes, including cancer, neurodegenerative diseases, and infectious diseases. The study of EVs is an active area of research, with ongoing efforts to develop standardized methods for their isolation, characterization, and functional analysis. The International Society for Extracellular Vesicles was established to coordinate research and standardize the field.