Nanoparticle Tracking Analysis (NTA) is a valuable technique for characterizing extracellular vesicles (EVs), providing information on size, size distribution, and concentration. The light-scattering mode of NTA offers accurate size and concentration data, useful for comparing EV isolation methods and storage conditions. The fluorescent mode of NTA allows differentiation of EV subgroups based on specific markers, making it effective for disease detection in biological fluids. However, the success of fluorescent NTA depends on the quality of fluorescent tags and labeling methods. NTA has limitations in differentiating EVs from other nanoparticles, highlighting the need for fluorescent NTA. The technique is crucial for EV quantification and standardization, with applications in disease diagnosis and therapy. Despite challenges in sample preparation and instrument calibration, NTA remains a powerful tool for EV characterization, offering enhanced specificity and sensitivity. Future research should focus on improving NTA's accuracy and expanding its applications in EV studies.Nanoparticle Tracking Analysis (NTA) is a valuable technique for characterizing extracellular vesicles (EVs), providing information on size, size distribution, and concentration. The light-scattering mode of NTA offers accurate size and concentration data, useful for comparing EV isolation methods and storage conditions. The fluorescent mode of NTA allows differentiation of EV subgroups based on specific markers, making it effective for disease detection in biological fluids. However, the success of fluorescent NTA depends on the quality of fluorescent tags and labeling methods. NTA has limitations in differentiating EVs from other nanoparticles, highlighting the need for fluorescent NTA. The technique is crucial for EV quantification and standardization, with applications in disease diagnosis and therapy. Despite challenges in sample preparation and instrument calibration, NTA remains a powerful tool for EV characterization, offering enhanced specificity and sensitivity. Future research should focus on improving NTA's accuracy and expanding its applications in EV studies.