Without argument, the most essential aspect of discerning the fundamental biochemistry, development of clinical applications, or the biotherapeutic implementation of extracellular vesicles (EVs) lies in the ability to efficiently and cost-effectively isolate those vesicles from their host media with high yields and purity. This general challenge is exacerbated by the fact that EVs, and more specifically exosomes, must be isolated from an extremely wide diversity of matrices, including mammalian body fluids (serum, urine, saliva, breast milk), plant materials, and a wide range of cell culture media. A final challenge lies in the fact that the sample/matrix volumes can range from microliters to milliliters, and even liters based on the application at hand; covering fundamental studies, clinical diagnostics, and gene therapy vector production. Described in this chapter are developments from the Clemson University laboratory in the use of capillary-channeled polymer (C-CP) fibers as stationary phases for the isolation of exosomes via a hydrophobic interaction chromatographyHydrophobic interaction chromatography (HIC) elution method. The novel-shaped fibers, when packed into a column format, provide 1–4 μm open channels for efficient solution flow and exosome interaction with the modestly hydrophobic polyester (PET) and nylon-6 fiber surfaces. The general strategy involves an injection of the sample into a concentrated salt solution (2 M (NH4)2SO4), wherein matrix salts, small ionic compounds, and genetic material from the sample matrix pass directly through the column or spin-down tip structures. Proteins initially adsorbed to the fiber surfaces are eluted through a reduction in the salt content and the addition of small percentages of organic solvent. Adsorbed exosomes are ultimately released with complete removal of salt content and a further increase in the organic solvent strength. C-CP fiber columns and spin-down tips provide high sample throughput on standardStandards high performance liquid chromatography platforms or through small-format (tabletop) centrifuge processing. Specific applications and results are presented across a wide diversity of sample matrices, suggesting distinct advantages relative to more commonly applied EV isolation methods. Very high throughput and separation quality combine with comparatively inconsequential materials costs to offer practical solutions to challenging exosome separations.