We describe the protocol development and optimization of asymmetric flow field-flow fractionation (AF4) technology for separating and characterizing extracellular nanoparticles (ENPs), particularly small extracellular vesicles, known as exosomes, and even smaller novel nanoparticles, known as exomeres. This technique fractionates ENPs based on hydrodynamic sizes and demonstrates a unique capability to separate nanoparticles with sizes ranging from a few nanometers to undefined level of micrometers. ENPs are resolved by two perpendicular flows, channel flow and cross flow, in a flat thin channel with a semi-permissive bottom wall membrane. The AF4 separation method offers several advantages over other isolation methods for ENP analysis, including being label-free, gentle, rapid (< 1 hour), and highly reproducible, and providing efficient recovery of analytes. Most importantly, in contrast to other available techniques, AF4 can separate ENPs at high resolution (1 nm) and provide a large dynamic range of size-based separation. In conjunction with real-time monitors, such as ultraviolet absorbance and dynamic light scattering, and an array of post-separation characterizations, AF4 facilitates the successful separation of distinct subsets of exosomes and the identification of exomeres. Though the whole procedure of cell culture and ENP isolation from the conditioned media by ultracentrifugation can take approximately three days, the AF4 fractionation step takes only one hour to perform. Users of this technology will require expertise in the working principle of AF4 to operate and customize protocol applications. AF4 can contribute to the development of high-quality, exosome- and exomere-based molecular diagnostics and therapeutics.