Plasma vitamin C (ascorbic acid + dehydroascorbic acid) concentration is a good index of the nutritional status of vitamin C. However, the methodologies for storage and analyses have not been investigated in bovine plasma. The validity of an analytical method for bovine plasma using high performance liquid chromatography (HPLC) with a spectrophotometric detector was examined. Exogenous dehydroascorbic acid was almost completely converted to ascorbic acid during the preparation for analysis with a reducing reagent, dithioerythritol. The analytical recoveries of ascorbic acid were high. Ascorbic acid was not detected after treatment with ascorbic acid oxidase. Thus, the specificity of this method is considered to be high. Although vitamin C was stable in plasma treated by dithioerythritol at −20°C for 6 days, vitamin C in untreated plasma significantly decreased during 3‐day storage at −20°C. These results indicate that the HPLC method is suitable for the determination of plasma vitamin C in cattle and that the storage conditions are important for determination of plasma vitamin C. Plasma vitamin C concentration ranged between 1.49 mg/L and 3.33 mg/L in fattening cattle. This result suggests that fattening cattle show large individual variation in plasma vitamin C concentration.