The majority, if not all, of human cell types secrete extracellular vesicles (EVs) into their environment, at least partly as a means of intercellular communication. These secreted vesicles can be detected in most bodily fluids including blood, urine, and saliva. The number of secreted vesicles and their composition is altered in various pathological conditions, raising opportunities to exploit EVs as diagnostic and/or prognostic biomarkers. For this to become a reality, it is important to reach consensus regarding the standardization of protocols for sample collection, EV isolation, handling, and storage for valid comparison and interpretation of measurements. Depending on the information required, there are several detection options including EV number and size distribution, molecular surface markers, procoagulation activity, and RNA content. For these purposes, different techniques are currently utilized or under development. This review discusses the techniques that have the potential to become standard EV detection methods in a clinical diagnostic setting. In addition to the accuracy of the detection technique, other factors such as high-throughput, cost-effectiveness, time consumption, and required operator skill are important to consider. A combination of increasing fundamental knowledge, technological progress, standardization of sample collection, and processing protocols is required for EVs to become reliable predictors of altered physiology or development of disease suitable for routine clinical diagnostics. Cancer and (cardio)vascular disorders are examples of pathologies where EV detection may be applied in the near future for diagnosis and/or prognosis.