Vitamin E comprises a group of compounds possessing vitamin E activity. alpha-Tocopherol is the compound demonstrating the highest vitamin E activity, which is available both in its natural form as RRR-alpha-tocopherol isolated from plant sources, but more common as synthetically manufactured all-rac-alpha-tocopherol. Synthetic all-rac-alpha-tocopherol consists of a racemic mixture of all eight possible stereoisomers. Assessing the correct biological activity in form of bioavailability and biopotency has been a great challenge during many years as it is difficult to measure clinical endpoints in larger animals than rats and poultry. Thus, the biological effects in focus are resorption of fetuses, testicular degeneration, muscle dystrophy, anemia, encephalomalacia, and in recent years the influence of vitamin E on the immune system are the most important clinical markers of interest. For humans and animals, only different biomarkers or surrogate markers of bioactivity have been measured. In studies with rats, a good consistency between the classical resorption-gestation test and the bioavailability of the individual stereoisomers in fluids and tissues has been shown. For humans and other animals, only different biomarkers or surrogate markers of bioactivity have been measured, and due to the lack of good biological markers for bioactivities, bioavailability is often used as one of the surrogate markers for bioactivities with those limitations this must give. Therefore, a relatively simple analytical method, which allows analysis of the individual stereoisomers of alpha-tocopherol, is an important tool in order to quantify relative bioavailability of the individual stereoisomers. The analytical method presented here allows the quantification of total tocopherol content and composition by normal phase HPLC and subsequent separation of the stereoisomers of alpha-tocopherol as methyl ethers by chiral HPLC. Using this method, the alpha-tocopherol stereoisomers are separated into five peaks. The first peak consists of the four 2S isomers (SSS-, SSR-, SRR-, SRS-), the second peak consists of RSS-, the third peak consists of RRS-, the fourth peak consists of RRR-, and the fifth peak consists of RSR-alpha-tocopherol. The discussion on the bioavailability of RRR- and all-rac-alpha-tocopheryl acetate has primarily been based on human and animal studies using deuterium-labeled forms, whereby a higher biopotency of 2:1 (of RRR: all-rac) has been demonstrated, differing from the accepted biopotency ratio of 1.36:1. In agreement with previous studies, the 2S-forms exert very little importance for the vitamin E activity due to their limited bioavailability. We find notable differences between animal species with regard to the biodiscrimination between the 2R-forms. Especially, cows preferentially transfer RRR- alpha-tocopherol into the milk and blood system. The distribution of the stereoisomer forms varies from tissue to tissue, and in some cases, higher levels of the synthetic 2R-forms than of the RRR-form are obtained, for example, for rats. However, the biodiscrimination of the stereoisomers forms is influenced by other factors such as age, dietary levels, and time after dosage. More focus should be given on the bioactivity of the individual 2R-forms rather than just the comparison between RRR- and all-rac-alpha-tocopheryl acetate.