Vitamin D₃ was resolved and quantified by applying reserved-phase high pressure liquid chromatography (RP-HPLC) to the samples of hen eggs. The samples were separated on a reserved-phase C₁₈ column with acetonitrile+methylenechloride (0.001% triethylamine)+methanol (700+300+0.5) as the mobile phase. Quantitative determination was accomplished by monitoring the ultraviolet absorbance of vitamin D₃ at 280 nm. The recovery was 94.43% for vitamin D₃ Details of the accuracy and precision of the method were presented.