MISEV2023 is an updated guide to extracellular vesicle (EV) research and applications, designed to serve as a comprehensive resource for both beginners and experienced researchers. The document covers a wide range of topics, including historical perspectives, nomenclature, collection and pre-processing, separation and concentration, characterization, release and uptake, functional studies, and in vivo studies. It emphasizes the importance of pre-analytical variables and provides detailed information on key approaches and techniques. The development of MISEV2023 involved extensive community input, with contributions from over 100 authors and nearly 700 co-authors. The process began with a pre-drafting survey in 2020, followed by multiple drafts, refinements, and rounds of feedback from the ISEV board and the community. The final version was formally approved for publication on December 13, 2023. MISEV2023 acknowledges its limitations, such as the complexity and length of the development process, and emphasizes that it should not be seen as an exhaustive review of the literature. The authors encourage critique and debate to improve the document and plan for future updates, considering more structured approaches and the use of new tools like artificial intelligence. The community has been actively promoting MISEV2023 through various channels, including editorials, journal clubs, and scientific meetings. The ultimate goal is to guide the EV community in advancing basic biology and translating findings into benefits for human health and broader applications.MISEV2023 is an updated guide to extracellular vesicle (EV) research and applications, designed to serve as a comprehensive resource for both beginners and experienced researchers. The document covers a wide range of topics, including historical perspectives, nomenclature, collection and pre-processing, separation and concentration, characterization, release and uptake, functional studies, and in vivo studies. It emphasizes the importance of pre-analytical variables and provides detailed information on key approaches and techniques. The development of MISEV2023 involved extensive community input, with contributions from over 100 authors and nearly 700 co-authors. The process began with a pre-drafting survey in 2020, followed by multiple drafts, refinements, and rounds of feedback from the ISEV board and the community. The final version was formally approved for publication on December 13, 2023. MISEV2023 acknowledges its limitations, such as the complexity and length of the development process, and emphasizes that it should not be seen as an exhaustive review of the literature. The authors encourage critique and debate to improve the document and plan for future updates, considering more structured approaches and the use of new tools like artificial intelligence. The community has been actively promoting MISEV2023 through various channels, including editorials, journal clubs, and scientific meetings. The ultimate goal is to guide the EV community in advancing basic biology and translating findings into benefits for human health and broader applications.