Extracellular vesicles (EVs) are nanoscale membrane-bound structures released by cells, serving as important biomarkers for disease diagnosis and intercellular communication. Despite their clinical potential, the lack of sensitive and efficient analytical technologies for EVs has hindered their translation to clinical use. This review discusses recent advances in EV analysis, focusing on new technologies for EV detection, characterization, and clinical applications. EVs are classified into exosomes, microvesicles, and apoptotic bodies based on their biogenesis. Exosomes are formed through endosomal pathways, while microvesicles are derived from plasma membrane blebbing. EVs contain a variety of molecular cargos, including proteins, lipids, and nucleic acids, and are enriched with specific markers such as tetraspanins and integrins. Physical characterization of EVs is achieved through techniques like electron microscopy, atomic force microscopy, and dynamic light scattering. These methods provide insights into EV size, morphology, and distribution. EV enrichment methods include ultracentrifugation, gradient centrifugation, co-precipitation, size-exclusion chromatography, and microfluidic filtering. These methods aim to isolate EVs from complex biological fluids while minimizing contamination. Protein analysis of EVs involves techniques such as western blotting, ELISA, and mass spectrometry, which help identify and quantify EV-associated proteins. Newer technologies, including flow cytometry, micro-nuclear magnetic resonance, and nano-plasmonic exosome (nPLEX) sensors, offer improved sensitivity and specificity for EV detection. Additionally, integrated magnetic-electrochemical exosome (iMEX) sensors enable rapid, point-of-care EV analysis. These advancements highlight the growing importance of EVs as biomarkers and therapeutic targets in disease diagnosis and treatment.Extracellular vesicles (EVs) are nanoscale membrane-bound structures released by cells, serving as important biomarkers for disease diagnosis and intercellular communication. Despite their clinical potential, the lack of sensitive and efficient analytical technologies for EVs has hindered their translation to clinical use. This review discusses recent advances in EV analysis, focusing on new technologies for EV detection, characterization, and clinical applications. EVs are classified into exosomes, microvesicles, and apoptotic bodies based on their biogenesis. Exosomes are formed through endosomal pathways, while microvesicles are derived from plasma membrane blebbing. EVs contain a variety of molecular cargos, including proteins, lipids, and nucleic acids, and are enriched with specific markers such as tetraspanins and integrins. Physical characterization of EVs is achieved through techniques like electron microscopy, atomic force microscopy, and dynamic light scattering. These methods provide insights into EV size, morphology, and distribution. EV enrichment methods include ultracentrifugation, gradient centrifugation, co-precipitation, size-exclusion chromatography, and microfluidic filtering. These methods aim to isolate EVs from complex biological fluids while minimizing contamination. Protein analysis of EVs involves techniques such as western blotting, ELISA, and mass spectrometry, which help identify and quantify EV-associated proteins. Newer technologies, including flow cytometry, micro-nuclear magnetic resonance, and nano-plasmonic exosome (nPLEX) sensors, offer improved sensitivity and specificity for EV detection. Additionally, integrated magnetic-electrochemical exosome (iMEX) sensors enable rapid, point-of-care EV analysis. These advancements highlight the growing importance of EVs as biomarkers and therapeutic targets in disease diagnosis and treatment.