This document specifies a method for the determination of the content of the total vitamin A (retinol), vitamin E (±-tocopherol) and vitamin D3 (cholecalciferol) content in animal feed using solid phase extraction (SPE) clean-up and high-performance liquid chromatography (HPLC). NOTE 1 The procedure enables also determination of vitamin D2 but with the use of another internal standard. The method is fully validated only for vitamin D3. The method has been successfully tested in collaborative trial for complete feed for broilers, pigs, and turkey, for premixture for broilers and piglets, for complementary feed for cows and mineral feed within the following ranges: vitamin A: 4 365 IU/kg 4 118 352 IU/kg; vitamin E: 22 mg/kg 13 800 mg/kg vitamin D3: 1 668 IU/kg 1 638 150 IU/kg. Quantification limits of 1 100 IU for vitamin A/kg (using UV-detection), 4 mg for vitamin E/kg (using UV-detection), 2 mg for vitamin E/kg (using fluorescence detection) and 2 000 IU for vitamin D/kg (using UV-detection) should be normally achieved. NOTE 2 The limits of quantification are minimum limits which were not determined within the validation study. Lower limits of quantification are possible but is validated by the user.