Hydrophobic interaction electrokinetic chromatography (HIEKC) was evaluated for the separation of nonionic hydrophobic pharmaceuticals utilizing vitamin A palmitate, vitamin E acetate, and vitamin D₃ as model compounds. In order to effectively dissolve the highly hydrophobic vitamins, the separation medium consisted of acetonitrile-water (80:20, v/v). Tetradecylammonium (TDA⁺) at a concentration of 80 mM was added to the separation medium as pseudostationary phase, and the three vitamins were separated prior to the electroosmotic flow based on different hydrophobic interactions to the charged TDA⁺ molecules. With a 47 cmx50 μm internal diameter uncoated fused-silica capillary, the three vitamins were baseline separated within 6 min utilizing a separation voltage of 10 kV. Migration times were repeatable within 1.3% and varied less than 4.2% between 5 different days. Peak areas were repeatable within 1.7% and varied less than 4.2% between 5 different days.