Extracellular vesicles (EVs) are membrane-bound nanoparticles that are secreted by most cell types with an emerging role in cellular communication and potential as biomarkers of disease. Nanoparticle tracking analysis (NTA) is a commonly used technique to measure the size and concentration of nanoparticles, such as EVs. Here, we present two protocols for the analysis of size profile concentration, and zeta potential (ZP) of well-characterized EVs derived from human choriocarcinoma JAr cells using NTA. These protocols describe how the size profile concentration, and ZP of JAr EVs are measured using optimized settings of NTA. With good experimental practices and consistent protocol, NTA measurements of EVs can provide reliable data that could potentially translate further uses of EVs for diagnostic and therapeutic applications.