The article reviews the role of tetraspanins in the formation and function of extracellular vesicles (EVs). Tetraspanins are abundant membrane proteins that form tetraspanin-enriched microdomains (TEMs) and play crucial roles in various biological processes. EVs, including microvesicles, exosomes, and apoptotic bodies, are released by many cell types and can transfer functional proteins, lipids, and RNAs between cells. Exosomes, in particular, have gained attention due to their potential as biomarkers and therapeutic agents. Tetraspanins are highly enriched in EVs and are involved in EV biogenesis, cargo selection, and target cell uptake. They organize TEMs, which serve as specialized platforms for the compartmentalization and sorting of proteins and RNA. Tetraspanins also regulate the targeting and uptake of EVs by recipient cells, influencing immune responses and disease progression. The article highlights the importance of tetraspanins in EV functions and their potential as therapeutic targets.The article reviews the role of tetraspanins in the formation and function of extracellular vesicles (EVs). Tetraspanins are abundant membrane proteins that form tetraspanin-enriched microdomains (TEMs) and play crucial roles in various biological processes. EVs, including microvesicles, exosomes, and apoptotic bodies, are released by many cell types and can transfer functional proteins, lipids, and RNAs between cells. Exosomes, in particular, have gained attention due to their potential as biomarkers and therapeutic agents. Tetraspanins are highly enriched in EVs and are involved in EV biogenesis, cargo selection, and target cell uptake. They organize TEMs, which serve as specialized platforms for the compartmentalization and sorting of proteins and RNA. Tetraspanins also regulate the targeting and uptake of EVs by recipient cells, influencing immune responses and disease progression. The article highlights the importance of tetraspanins in EV functions and their potential as therapeutic targets.