Extracellular vesicles (EVs), including exosomes and microvesicles, have been shown to carry a variety of biomacromolecules including mRNA, microRNA and other non-coding RNAs. Within the past 5 years, EVs have emerged as a promising minimally invasive novel source of material for molecular diagnostics. Although EVs can be easily identified and collected from biological fluids, further research and proper validation is needed in order for them to be useful in the clinical setting. In addition, innovative and more efficient means of nucleic acid profiling are needed to facilitate investigations into the cellular and molecular mechanisms of EV function and to establish their potential as useful clinical biomarkers and therapeutic tools. In this article, we provide an overview of recent technological improvements in both upstream EV isolation and downstream analytical technologies, including digital PCR and next generation sequencing, highlighting future prospects for EV-based molecular diagnostics.