Extracellular vesicles (EVs) are small vesicles ensuring transport of molecules between cells and throughout the body. Their small size and biological and physical functions make them optimal candidates as therapeutic agents in several medical frameworks. The elucidation of biophysical and biochemical characteristics of EVs is crucial for the understanding of their interaction with recipient cells and their functional activity. In particular, for therapeutic applications understanding how the process of manufacturing affects the biological function of EVs is mandatory before going to the clinical testing. The absence of standardized methodologies and technologies to establish reliable criteria has been the main hurdle for real therapeutic applications of EVs. Here we exploited a combination of different biophysical techniques (Fourier Transform Infrared Spectroscopy, Ultraviolet Resonant Raman Spectroscopy, Atomic Force Microscopy and Small Angle X-Ray Scattering) to screen EVs derived from umbilical cord-mesenchymal stem cells with different isolation procedures, evidencing subtle differences in the biophysical properties of EVs. The results underline the need of a multiparametric analysis to address size, stability and purity of different preparations and to correlate phenotypic parameters of EVs with their functional activity.