Abstract: An isocratic RP-HPLC method for the determination of the related substances of vitamin D₃ in its preparations was established.The chromatographic separation was achieved on amethyst C₁₈-H column (150 mm×4.6 mm，5 μm），using acetonitrile as the mobile phase.The detection was performed at 265 nm，and the column temperature was 20 °C.A good resolution with baseline separation was obtained.Impurities were identified using photochemical and thermal experiments and LC-MS-DAD.The APCI-MS spectra gave the same M+H⁺ peak at m/z 385.27 as their base peaks for vitamin D₃，pre-vitamin D₃，trans-vitamin D₃，and tachysterol ₃.The UV λ_max of vitamin D₃,pre-vitamin D₃,trans-vitamin D₃,and tachysterol3 were 265 nm，265 nm，275 nm and 279 nm，respectively.The method was specific and robust.The limit of detection was 0.1 μg/mL，and the limit of quantification was 0.3 μg/mL.Test solution was stable for 24 h at room temperature.Three kinds of vitamin D₃ preparations were tested using the developed method and the current official method，the results showed no differences.The developed method was suitable for the determination of the related substances of vitamin D₃ in its preparations.