Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology. Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).