Exosome characterization by nanoparticle tracking analysis (NTA) is a cutting-edge technique widely used in the study of extracellular vesicles. Exosomes, small membrane-bound particles secreted by cells, play critical roles in cell communication and are implicated in various physiological and pathological processes, including cancer, cardiovascular diseases, and neurological disorders. Nanoparticle tracking analysis provides a dynamic method for measuring the size distribution and concentration of exosomes in a sample by visualizing and tracking their movement under a laser beam. This technology is particularly valuable because it offers real-time analysis at the nanoscale, allowing researchers to obtain precise and quantitative data on exosome populations. By utilizing NTA for exosome characterization, scientists can investigate the heterogeneity of exosome samples, understand their biogenesis, and decipher their functional roles in disease. This information is instrumental in the development of exosome-based diagnostics and therapeutics, advancing our capabilities to harness these nano-sized vesicles for medical applications and improving our understanding of their contribution to health and disease.