This International Standard specifies a method for the determination of vitamin D in a test sample containing at least 10 μg of vitamin D per 100 g [equal to 400 International Units (IU) of vitamin D per 100 g] by using high-performance liquid chromatography (HPLC). The results of the determination are only reliable if, in the case of determination of vitamin D3, the test sample contains only vitamin D3, and no vitamin D2 (which will be added as an internal standard) and, in the case of determination of vitamin D2, the test sample contains only vitamin D2 and no vitamin D3 (which will be added as an internal standard). This is to be verified by the procedure carried out without the addition of the internal standard (vitamin D2).