The study of extracellular vesicles (EVs) has evolved at a rapid rate over the past few years. The knowledge concerning EV origin, their role(s) in cell biology, and their molecular cargo have contributed to a deeper understanding of EVs’ biology, which is facilitating their use as important clinical-based biomarkersBiomarkers. Much attention has been given to the importance of nucleic acids, especially a plethora of different ribonucleic acids (RNA), namely total RNA (TRNA), packaged into EVs. The immense EV research progress has been realized through efforts in developing novel technologies for EV isolation and the subsequent analysis of their low abundant cargo, including TRNA. In this chapter, we will discuss isolation technologies of EVs and the analysis of their RNA cargo, and highlight the specific challenges associated with conducting these studies. In addition, we will provide information on the differences in RNA content within EVs to that found in biological cells. Finally, we will present examples using EV-TRNA in managing a number of different disease states.