Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different biological molecules, including protein, lipid, DNA, and RNA, and are postulated to facilitate cell-to-cell communication in diverse tissues and organs. Recently, EVs have attracted significant attention as biomarkers for diagnostics and therapeutic agents for various diseases. Many methods have been developed for EV characterization. However, current methods for EV analysis all have different limitations. Thus, developing efficient and effective methods for EV isolation and characterization remains one of the crucial steps for this cutting-edge research field as it matures. Here, we provide a detailed protocol outlining a single-particle interferometric reflectance imaging sensor (SP-IRIS), as a method that is capable of detecting and characterizing EVs from unpurified biological sources and purified EVs by other methodologies. This advanced technique can be used for multi-level and comprehensive measurements for the analysis of EV size, EV count, EV phenotype, and biomarker colocalization.