Body fluids contain large amounts of submicron (majority <200nm) extracellular vesicles (EVs) originating from a variety of cell-types. Cells concurrently release different types of EVs, and because the molecular composition of EVs depends on the status of the releasing cell, EVs are not only very abundant, but also very heterogeneous in appearance and composition. EVs play a role in homeostasis and intercellular communication in biological systems and EV-based biomarkers are an emerging field for clinical application. Consequently, the global and broad interest in detection and extraction of information from EVs is increasing rapidly and comprises both basic biology-driven research and clinical research. Unfortunately, most commonly used techniques to identify and characterize EVs either lack the sensitivity and specificity for single EV-based analysis (e.g. conventional flow cytometry (detection limit of ~300 nm EVs)) or are low-throughput (e.g. electron microscopy, atomic force microscopy). Hence we developed a high-resolution fluorescence-based flow cytometric method enabling integrated analysis of multiple parameters such as light scattering, quantity, buoyant density and surface markers (based on antibody binding) of individual submicron EVs. This technique appeared to be very useful for simultaneous quantitative and qualitative analysis of large numbers of EVs, a prerequisite for rare event analysis, e.g. analysis of circulating tumor-cell derived EVs. Using this technology we showed heterogeneity in immune cell-derived nano-sized EVs and demonstrated the active control of EV-subset release and composition. Recently, we established a protocol to physically sort specific EV subsets thereby opening novel possibilities for comprehensive molecular characterization of isolated EV subsets.