Extracellular vesicles (EVs) are membrane-enclosed particles that have gained significant attention for their potential in disease diagnosis and therapy. However, the clinical translation of EVs is hindered by their heterogeneity and inconsistent dosing. Nanoparticle tracking analysis (NTA) is a valuable technique for characterizing EVs, offering insights into size, size distribution, concentration, and protein markers. This review discusses the NTA technique, focusing on factors affecting results and its two modes: light-scattering and fluorescent. The light-scattering mode provides accurate size, size distribution, and concentration information, while the fluorescent mode allows for the differentiation of EV subgroups based on specific markers. The success of fluorescence NTA depends on fluorescent tags and labeling methods. When EVs are labeled with disease-specific markers, fluorescence NTA offers an effective tool for disease detection in biological fluids. The review also highlights the limitations and future directions of NTA in EV characterization, emphasizing the need for standardization and further technological advancements.Extracellular vesicles (EVs) are membrane-enclosed particles that have gained significant attention for their potential in disease diagnosis and therapy. However, the clinical translation of EVs is hindered by their heterogeneity and inconsistent dosing. Nanoparticle tracking analysis (NTA) is a valuable technique for characterizing EVs, offering insights into size, size distribution, concentration, and protein markers. This review discusses the NTA technique, focusing on factors affecting results and its two modes: light-scattering and fluorescent. The light-scattering mode provides accurate size, size distribution, and concentration information, while the fluorescent mode allows for the differentiation of EV subgroups based on specific markers. The success of fluorescence NTA depends on fluorescent tags and labeling methods. When EVs are labeled with disease-specific markers, fluorescence NTA offers an effective tool for disease detection in biological fluids. The review also highlights the limitations and future directions of NTA in EV characterization, emphasizing the need for standardization and further technological advancements.