The importance of exosomes and other extracellular vesicles (EVs) in cell–cell communication cannot be overstated. Thus, many efforts are being invested in intercepting exosomes in order to analyze their content and gain information both on the cells that produce them and on the cells that receive them. The holy grail of EV research is to associate specific EV biomarkersBiomarkers with the tissues that produced them, as this will open endless opportunities for diagnostics. One of the challenges in this type of research is to identify different subpopulations within a biological sample, which can be addressed using a comprehensive analysis at a single exosome level. Single-molecule localization microscopyMicroscopy (SMLM) is a term used to describe several super-resolution microscopyMicroscopy methods, all of which are based on the common principle of identifying the precise localization of individual fluorophores that are usually associated with a target of interest. SMLM is based on the ability of certain fluorophores to stochastically switch from an OFF, dark state, to an ON, bright state, so that at any given moment only some of the fluorophores are ON while others are OFF. Temporally resolving nearby fluorophores allows scientists to obtain a resolution of ≥20 nm, well within the range of single exosomes. By analyzing thousands of individual exosomes, you gain strong statistical confidence and the ability to detect even rare subpopulations. In this chapter, we discuss the unique features of SMLM in exosome research and we provide recent examples where these cutting-edge methods were applied in diagnostics and therapeutics.