Hesperidin (2S-form), the flavanone 7-O-glycoside, is the main constituent of some Citrus species. The peels of two Citrus species are used as a crude drug, Aurantii nobilis pericarpium, in the Japanese Pharmacopoeia and as components in Kampo formulae. Thus, HPLC analysis of hesperidin as a marker compound is needed for quality control of medicines. Hesperidin was separated from the corresponding C-2 epimer by normal-phase HPLC using a chiral column. Moreover, narirutin and neohesperidin were also separated from the corresponding C-2 epimer. The analyses of commercial hesperidin samples revealed that they contained the C-2 epimer and that the relative ratio of hesperidin to the epimer ranged from 92:8 to 59:41. The HPLC application to Citrus extracts suggested that naturally occurring hesperidin in Citrus has the 2S configuration; however, the dry extracts of rikkunshito and chotosan, which are Kampo formulations containing Aurantii nobilis pericarpium, were found to contain a considerable amount of the (2R)-epimer. These data suggest that the decoction process of the formulae partly converts hesperidin to the epimer. Because diastereomers differ from each other in physicochemical and biological activities, HPLC to separate hesperidin from the C-2 epimer should be introduced into the letter of approval for herbal medicines.