Abstract: In this study, an HPLC-DAD method with high separation efficiency, detection sensitivity, and selectivity was developed and validated for the simultaneous qualitative and quantitative determination of anthraquinones (aloe-emodin, rhein, emodin, chrysophanol, and physcion) in Rumex acetosella L. and Rumex crispus L. plants collected at different regions and times. Optimal method conditions were determined by studying different mobile phases, mobile phase compositions, flow rates, column, column temperatures, injection volume, and wavelength of the compounds. Validation studies were carried out to prove the validity of the developed method. The correlation coefficients for all compounds were found to be in the range of 0.9991–0.9997, and the calibration curves were linear in the concentration range of 0.1–1.0 μg/mL. The limits of detection and quantification values were calculated in the range of 0.014–0.017 and 0.017–0.057 μg/mL, respectively. The developed and validated method indicated that it is effective and suitable for determining aloe-emodin, rhein, emodin, chrysophanol, and physcion in extracts of Rumex plants. In addition, the antioxidant activities of the plant extracts were determined using the Cupric Ion Reducing Antioxidant Capacity and 2,2-diphenyl-1-picrylhydrazil radical assays, revealing their antioxidant properties.