Abstract: Aim To establish a sensitive and specific HPLC method for controlling the quality of total glucosides from paeony (TGP). Methods HPLC method was applied for quality assessment of TGP. The preparation of sample, the HPLC column, mobile phase, elution mode (isocratic or gradient) and gradient program were optimized in order to obtain high quality HPLC profile. The HPLC system consisted of a Waters 600 pump and a 996 photodiode-array detector (DAD). Data were acquired and processed with the Millennium 2010 workstation. HPLC analysis was performed on a Supelcosil^TM LC-18 column (5 μm, 150 mm×4.6 mm) with the mixture of acetonitrile (A) and H₂O (acidified to pH 3.0 with phosphoric acid) (B) as mobile phase in gradient mode. The concentrations of solvent A were 10%, 15%, 18%, 30%, 35%, 40% and 40% at 0, 5, 25, 27, 38, 40 and 50 min, respectively. The column temperature was setup at 25 ℃ and the flow-rate was 1 mL·min^-1. The reference solution of chemical standards and sample were injected into HPLC system, respectively. Results The HPLC chromatographic fingerprinting of TGP,showing 22 characteristic peaks,was established from 10 lots of TGP products. These peaks could be divided into 3 groups: From 1 to 3 min, there are 3 low peaks. From 5 to 20 min, there are 8 peaks including the main diagnostic peaks 5, 6 and 7. By comparison of the retention time and the on-line UV spectra of chemical standards, peak 5 and 6 were identified as albiforin and paeoniflorin, respectively. From 20 to 36 min, 11 peaks were observed, in which peak 12 was one of the main diagnostic peaks. The ratios of peak area between 5, 6, 7 and 12 were 0.267-0.348∶1∶0.108-0.135∶0.405-0.537. Moreover, comparison of the HPLC profiles of TGP and the roots of Paeonia lacttiflora Pall indicated that they were closely related to each other. Conclusion The chromatographic fingerprinting of TGP with high specificity can be used to control its quality and assure lot to lot consistency.