Extracellular vesicles (EVs) are natural-occurring lipid bilayer membrane structures secreted by cells. An increasing number of findings have shown the involvement of EVs in cell-to-cell communication, highlighting their pivotal role in transferring of information. They can carry both small molecules like metabolites, and macromolecules including proteins, nucleic acids and lipids. Interesting features of EVs include the ability in crossing biological barriers, stability in circulation and biological fluids, and precise-targeting delivery. Furthermore, they exhibit low toxicity and immunogenicity. Altogether, EVs are very attractive as possible nanocarriers, in both forms i.e., as they are and after biological engineering. Protocols regarding their isolation and characterization are constantly updated thanks to the improvement of types of equipment or innovative approaches used. The use of EVs as biomarkers and biological nanocarriers has various limitations. Attempts to load EVs with exogenous compounds are very recent and the evaluation of their biological effects require complex cellular models or specific protocols in animal models. In addition, no gold standard methods are currently established for the isolation and concentration of pure EVs from cell culture, tissue, and biological fluids. The aim of this Methods Collection is to deal with the current methods for isolation and characterization of EVs and includes protocols regarding both their use as nanocarriers (e.g., EVs-loading and functionalization methods) as well as their evaluation on biological models (e.g., in vitro cell model to evaluate EVs capability to cross biological barriers).