<p>Human embryonic kidney (HEK) cell lines are used in the production of diverse biopharmaceuticals, including extracellular vesicles (EVs), due to their scalability and human biocompatibility. EVs are nanosized (30–200 nm), carriers of biomolecules that participate in cellular communication and can deliver therapeutic cargo in a targeted manner. Their use as drug vectors requires high-purity isolates; however, achieving such purity is challenging due to complex host matrices. Size exclusion chromatography (SEC) affects separations based on hydrodynamic radii and is a common isolation method due to its downstream applicability; however, it struggles to yield high-purity isolates. Polyester (PET) capillary-channeled polymer (C-CP) fiber spin-down tips are a novel EV separation platform employing hydrophobic interaction chromatography (HIC) to isolate high-purity EVs. Samples are loaded under high ionic strength conditions, where hydrophobic species adsorb to the fibers, and polar species pass through unretained. Analytes are then eluted in order of increasing hydrophobicity using an inverse salt gradient and adding small amounts of organic solvent at each step. A practical comparison of a commercial SEC column and PET C-CP spin-down tip EV eluates is conducted here via quantitative absorbance response curves, protein assays, nanoflow cytometry, and transmission electron microscopy. PET C-CP fiber platforms isolated 4.3 × 10¹¹ particles mL⁻¹ of HEK supernatant and yielded purities of 3.1 × 10¹⁰ particles µg⁻¹ protein, a tenfold improvement for both metrics versus the SEC methodology, demonstrating a rapid, high-purity alternative to that commercial method.</p>