Anthraquinone derivatives are the major bioactive components in rhubarb.Due to lack of quantitative analysis method, a full quality control of rhubarb has been limited.A reverse phase ultra-high-performance liquid chromatography (UHPLC) analytical method was developed for simultaneous quantification of 10 anthraquinone derivatives in rhubarb.This study focused the influence of analytical parameters on separation efficiency of five anthraquinone glucosides in rhubarb.The UHPLC method was performed by a Waters Acquity BEH C18 column (2.1 mm × 100mm, 1.7μm) with binary gradient mobile phase consisting of a mixture of methanol and 0.1% (v/v) aqueous phosphoric acid.In order to obtain a robust chromatographic resolution of five anthraquinone glucosides, relatively important chromatographic parameters including retention time, resolution, theoretical plate number, symmetry factor were systematically investigated by altering column temperature and flow rate.The column temperature and flow rate have a non-linear relationship with theoretical plate number and symmetry factor;the optimal column temperature was 30 ℃ and the optimal flow rate was 0.20 mL/min.A new UHPLC analytical method was developed based on these optimal parameters.This UHPLC-based analytical method was successfully applied to quantitative determination of 10 anthraquinone derivatives in Rheum tanguticum Maxim.ex Balf and Rheum palmatum L.There are remarkable content differences between aloe-emodin-8-o-β-D-glucoside (more than 15 times) and rhein-8-o-β-D-glucoside (more than 12 times) in the two species of rhubarb tested.Compared with the analytical method used for rhubarb in Chinese pharmacopoeia, the present method is more environmental friendly because toxic reagent (chloroform) is avoided and mild conditions are sufficient as no hydrochloric acid for sample preparation is used.In conclusion, the devised technique is suitable for quality control of rhubarb.