This study was carried out as a preliminary step for analyzing the vitamin D₂ and D₃ in feedstuffs using HPLC. 1. Absorbance curve following the wavelength of UV light showed that the 264㎚ gave a maximum absorbance. 2. The mixed solution of 20 ppm vitaminD₂ and D₃ was injected in HPLC with μ Bondapak C_(18) column. The vitamin D₂ was resolved at 14.8 min. and D₃ at 15.4 min. after injection when eluted with 95% methanol + 5 % H₂O. 3. Varying amount of vitamin D₂ was injected into the column of μ Bondapak C_(18), and the detector 240 showed that the least detectable level was 5ng and that 5-100ng range brought a linear calibration curve. 4. When μ Bondapak column and 440 detector were used, 10-100ng range gave a linear standard curve. 5. When μ Porasil column and 440 detector were applied, 10-100ng range showed a linear calibration curve.