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High-performance liquid chromatography coupled with photodiode array detection and evaporative light scattering detection (HPLC-DAD-ELSD) was established to determine paeoniflorin and albiflorin simultaneously in Radix Paeoniae Rubra. The assay was performed on a Diamonsil C18 (4.6 mm × 250 mm, 5 μm) column by a gradient elution program with acetonitrile and aqueous formic acid (0.05% v/v) as mobile phase at a flow rate of 1.0 mL min-1. The detection wavelength of DAD was 230 nm, and the evaporator tube temperature of ELSD was set at 110 °C with the nebulizing gas flow rate of 3 L min-1. The temperature of column was kept at 30 °C. The linear ranges of paeoniflorin and albiflorin were within 0.050-1.510 mg mL-1 and 1.007-5.035 mg mL-1. The recoveries of paeoniflorin and albiflorin were 96.2-102.9% and 95.0-102.4%, respectively, while the relative standard deviation (RSD) of them was 0.2-2.5%. This method was quick, simple, accurate, and specific. It could be used for the quality control of Radix Paeoniae Rubra. The proposed approach was expected as a powerful tool for the quality control of Radix Paeoniae Rubra. [ABSTRACT FROM AUTHOR]