A novel ultra-performance LC-photodiode array-el-ESI-MS/MS screening method was developed for the detection and identification of natural antioxidants from radix et rhizoma Rhei. Nine compounds were found to possess a potential antioxidant activity, and their free radical-scavenging capacities were investigated in detail. The nine compounds were identified as 1-O-galloyl-2-O-cinnamoylglucose, 6-hydroxymusizin-8-O-β-D-glucopyranoside, (+)-catechin, gallic acid 3-O-β-D-glucopyranoside, trans-3,5,4'-trihydroxystilbene- 4'-O-β-D-(2"-O-galloyl)-glucopyranoside, sennoside A, 4-(4'-hydroxyphenyl)-2- butanone-4'-O-β-D-(2"-O-galloyl-6"-O-p-coumaroyl) glucopyranoside, emodin-8-O-(6'-Omalonyl) glucopyranoside, and physcion-8-O-β-D-glucopyranoside. The reactivity and SC(50) values of those compounds were investigated, respectively. 1-O-Galloyl-2-O-cinnamoylglucose showed the strongest capability for scavenging 1,1-diphenyl-2-picrylhydrazylfree radical; trans-3,5,4'-trihydroxystilbene-4'-O-β-D-(2"-O-galloyl) glucopyranoside showed the strongest capability for scavenging superoxide radical; 4-(4'-hydroxyphenyl)-2-butanone- 4'-O-2-D-(2"-O-galloyl-6"-O-p-coumaroyl) glucopyranoside exhibited the highest reactivity in the lipid peroxidation processes. The use of the analytical screening method based on ultra-performance LC-photodiode array-el-ESI-MS/MS would provide a new way for rapid detection of radical-scavenging natural compounds from radix et rhizoma Rhei or complex matrices.