Abstract: Vitamin D2 and D3 either in capsules or in oil preparations were determined by RP-HPLC. The sample was extracted ultrasonically with methanol. The extract was diluted to 100 mL with methanol, which was used for HPLC analysis after filtering on 0.45 μm filtering membrane. The reverse phase chromatographic column Zorbax SB-C18 and the protective column C18 were used for separation with pure acetonitrile as mobile phase (flow rate 1.0 mL·min-1). UV detection at the wavelength of 265 nm was adopted in the determination. Volume of sample solution taken for introduction was 20 μL. Linear relationships between values of peak area and mass concentration of both Vitamin D2 and D3 were found in the same range of 0.50-20 mg·L-1. Detection limit (3S/N) of 0.030 mg·L-1 was found for Vitamin D2 and 0.026 mg·L-1 for Vitamin D3. This method was used in analysis of substancial samples and using these samples as matrixes, tests for recovery were made by standard addition method, values of recovery were found in the range of 95.0%-99.4%, with values of RSD′s (n=6) in the range of 1.2%-2.6%.