Extracellular vesicles (EVs) are nanosized particles excreted by cells, which are associated with various physiological and pathological functions. They play a key role in intercell communication and are used as transport vehicles for various cell components. State of the art analysis methods are not able to provide label free chemical information at the single vesicle level , hence, new analysis techniques are required to study the chemical difference within EV (sub-)population. We introduce a protocol to profile structure and composition of individual EVs with the help of photothermal scanning probe infrared spectroscopy (AFM-IR).