Objective To develop a simple, sensitive, and precise method for simultaneous determination of 10 anthraquinones in Rhubarb. Methods HPLC-Q-HR/MS was employed for simultaneous quantification of free anthraquinones(aloe-emodin, emodin, chrysophanol, physcion, and rhein) and their glycosides. Chromatographic analysis was performed on an XBridge~(TM) C_(18) column(2.1 mm × 150 mm, 5 μm) with mobile phases consisting of 3 mmol/L ammonium acetate(A) and methanol(B) at a flow rate of 0.3 mL/min. Results All calibration curves exhibited good linear relationship(R~2 > 0.999). The limits of detection(LOD) and quantification(LOQ) were in the range of0.39-2.97 ng/mL and 0.56-8.90 ng/mL, respectively. The overall intra-and inter-day precisions of analytes presented as relative standard deviations(RSDs) were less than 2.79%. Relative recoveries varied between 97.83% and 104.28%. The validated method was applied to assess the quality of Rhubarb collected from different regions of China. Results showed that chrysophanol and rhein-8-O-β-D-glucoside was the largest portion of free anthraquinones and anthraquinone glycosides in Rhubarb, respectively. The total content of anthraquinones was higher in Rhubarbs from Sichuan, Qinghai, Yunnan, and Gansu provinces than that in those from Shandong and Henan provinces, while no significant variability existed in different regions of the same province.Conclusion HPLC-Q-HR/MS method is accurate and reliable for simultaneous quantification of above free anthraquinones and their glycosides in Rhubarb and can be applied to standardize the quality of Rhubarb and its quality control in different regions. Methods HPLC-Q-HR / MS was employed for simultaneous quantification of free anthraquinones (aloe-emodin, emodin, chrysophanol, physcion, and rhein) and their glycosides. Chromatographic analysis was performed on an XBridge ~ (18) column (2.1 mm × 150 mm, 5 μm) consisting of 3 mmol / L ammonium acetate (A) and methanol a Flow rate of 0.3 mL / min. Results All calibration curves exhibited good linear relationship (R ~ 2> 0.999). The limits of detection (LOD) and quantification (LOQ) were in the range of 0.39-2.97 ng / mL and 0.56-8.90 ng / mL, respectively. The overall intra-and inter-day precisions of analytes presented as relative standard deviations (RSDs) were less than 2.79%. Relative recoveries varied between 97.83% and 104.28%. The validated method was applied to assess the quality of Rhubarb collected from different regions of China. Results sh owed that chrysophanol and rhein-8-O-β-D-glucoside was the largest portion of free anthraquinones and anthraquinone glycosides in Rhubarb, respectively. The total content of anthraquinones was higher in Rhubarbs from Sichuan, Qinghai, Yunnan, and Gansu provinces than that in those from Shandong and Henan provinces, while no significant variability existed in different regions of the same province. Conlusion HPLC-Q-HR / MS method is accurate and reliable for simultaneous quantification of free anthraquinones and their glycosides in Rhubarb and can be applied to standardize the quality of Rhubarb and its quality control in different regions.