Extracellular vesicles (EVs) and especially nanosized exosomes need time-effective, easy-to-operate, and easily automated isolation methods/techniques that are not expensive, provide a controlled yield of high purity, and preserve the integrity of the fragile isolates. In addition, the EV isolates should not be contaminated with lipoproteins or other proteins. Reliable isolation methods can help to understand further the function and behavior of EVs at a cellular level, and this information can potentially be beneficial for other applications, such as drug delivery, antibody-based therapeutics, and diagnostic tools. This chapter gives a comprehensive overview of the potential of immunochromatography (IAC) and immunocapture techniques that exploit antibodies with a specific affinity toward biomolecules on the EV membrane surface, and versatile and gentle field-flow fractionation (FFF) techniques with possibilities for on-line coupling systems for the isolation of EV subpopulations with further experimental analysis. Special attention is given to methodological, and technical challenges, and the achievements are demonstrated with reference to applications. In addition, the principle of the IAC and FFF techniques are introduced to facilitate their methodological understanding.