Extracellular vesicles (EVs) are small vesicles ensuring transport of molecules between cells and throughout the body. EVs contain cell-type specific signatures and have been proposed as biomarkers in a variety of diseases. Their small size and biological and physical functions make them optimal candidates for therapeutic agents in immune therapy, vaccination, regenerative medicine, and drug delivery. Indeed there is no objective set of criteria available for designing synthetic EVs for a specific task in biomedicine. It is therefore urgent and critical to address these issues for EVs-based medicine to fulfill its promise. Here, we try to assess the phenotypic properties of EVs, through a multi-technique characterization based on Dynamic Light Scattering (DLS), FTIR spectroscopy, Atomic Force Microscopy (TEM) and Small Angle X-Ray Scattering (SAXS). This detailed analysis allows us to model, visualize and quantify EVs’ physical and chemical properties up to single vesicle level and it serves as a basis for the correlation of phenotypic parameters of EVs with their functional activity. The proposed work could allow for the design of innovative strategies for their sorting and detection, and for the personalized nanomedicine in general.