Abstract A simple and rapid quantitative method is described for determining the presence and amount of vitamin D3 (cholecalciferol) in mineral feed premixes by high pressure liquid chromatography. The samples were extracted with dichloromethane–pentane–methanol (46+46+8) after treatment with ammonium hydroxide, cleaned up by silica Sep-pak, and analyzed by normal phase high pressure liquid chromatography: 10 μm LiChrosorb NH2 column, hexane-isopropanol (98+2) mobile phase, ultraviolet detection at 254 nm. Recoveries were better than 95% in the range 25 000–100 000 IU/kg.