The phenolic constituents in the roots of Rheum officinale and Rubia cordifolia were identified with the aid of high-performance liquid chromatography and liquid chromatography-mass spectrometry and by comparison with authentic standards. A total of 17 hydroxyanthraquinones, gallic acid, and tannins were separated, and 14 of them were identified, being the main phenolic constituents present. Their antioxidant activity (Trolox equivalent antioxidant capacity) was evaluated using the improved 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt method. Hydroxyanthraquinones were the predominant antioxidant phenolic constituents in the roots of R. cordifolia, and tannins and gallic acid were the predominant antioxidant phenolic constituents in the roots of R. officinale. The structure-radical scavenging activity relationships of the tested hydroxyanthraquinones were systematically demonstrated as follows: Hydroxy groups on one benzene ring of the anthraquinone structure were essential for hydroxyanthraquinones to show activity, the ortho-dihydroxy structure in the hydroxyanthraquinone molecules could greatly enhance their radical scavenging effect, and glycosylation of the hydroxyanthraquinones reduced activity.