A procedure for determining and separating a mixture of fat-soluble vitamins by gradient thin-layer chromatography was developed. It was shown that a theoretical approach could be used to select the optimum thin-layer chromatographic conditions for determining tocopherol acetate, retinol acetate, ergocalciferol, and beta-carotene. The developed procedure was validated in terms of detection limit, specificity, efficiency, and precision. The procedure could be used for quality control of complex multivitamin preparations, medicinal plant raw material, and vegetable oils, oil extracts, and dietary supplements based on them.