Natural compounds are a good substitute for synthetic antioxidants. Attempts have been made to characterize the antioxidant capacity of natural resources (e.g., medicinal plants). Thus, the Rheum emodi Wall was evaluated using liquid chromatography with diode array detection and electrospray ionization-mass spectrometry. Three antioxidant compounds (i.e., myricitrin, myricetin-3-galloyl rhamnoside, and myricetin) were isolated, identified, and used to screen the antioxidant capacity of the new compounds. 2,2-Diphenyl-1-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and superoxide dismutase assay results are presented in the half-maximal inhibitory concentration values ranging 1.50∼28.46, 102.01∼137.55, and 4.06∼15.74 g/mL, respectively. Myricetin had the highest antioxidant activity among the other compounds. A significantly positive correlation was noted between the ethyl acetate fraction and the antioxidant compound. In a partial least squares-discriminant analysis model, identified antioxidant compounds were shown to play a role in the structure of the compound and its contents based on the antioxidant activity. The study suggests that myricetin from R. emodi possesses the most potent antioxidant activity, and thus is the most efficient in extracting antioxidant contents.