ABSTRACTIntroductionPaeoniae radix is one of the most important crude drugs used in Kampo medicines (KMs). A part of its pharmaceutical properties is due to the presence of paeoniflorin (PF) and albiflorin (AF).ObjectiveFor the specific and easy identification of PF and AF, an immunostaining technique was developed using anti‐PF monoclonal antibody (MAb).MethodologyPF and AF were treated with a NaIO4 solution and reacted with bovine serum albumin (BSA) preparing PF‐ and AF‐BSA conjugates on the polyethersulphone (PES) membrane. Anti‐PF MAb was bound and then antibody labelled with peroxidase directed against anti‐PF MAb. Finally, a substrate was added and then PF and AF were detected.ResultsAnti‐PF MAb recognised not only PF but also AF when 10 µg was present on the PES membrane. As little as 0.5 µg of PF and AF were still detected under immunostaining. Various Paeoniae radix samples and KMs were qualitatively analysed, and total amounts of PF and AF were visually detected by immunostaining technique. This method was applied to investigate the distribution of PF and AF in fresh peony root using immunoblotting by transferred from peony root to the PES membrane.ConclusionThe technique permitted the visualisation of PF and AF on PES membrane using immunostaining. The immunostaining technique established would be a powerful tool for probing the sources of PF and AF in plant extracts. Copyright © 2012 John Wiley & Sons, Ltd.