This study was carried out to establish an analytical method of vitamin D₂ or D₃ in feedstuffs. The ladino clover and orchard grass hays, and calf grower ration were used as samples. The vitamin D₂ or D₃ solution was added into the samples as an internal standard. The sample and vitamin D were saponified, extracted, and cleaned up through alumina, polyethylene glycol 600-Chromosorb W and florex columns. Then Sep-Pak silica and C_(18) cartridges were utilized for further purification. The Sephadex LH-20 column and μ Hondapak C_(18) column (HPLC, reverse phase) were applied to remove the remained impurities. Finally the normal phase μ Porasil column HPLC was utilized to quantitate the vitamin. It was known that the mean recovery of internal standard was 68% (S.D. 6%). The vitamin D₂ contents in ladino clover and orchard grass hays were 1,280±83 and 1,324±79 IU/㎏, respectively. The vitamin D₃ in calf grower ration was 1,463±102 IU/㎏.