A reversed-phase high-performance liquid chromatography (RP-HPLC) method with diode array detection has been developed for analysis of the major polyphenols in the roots and rhizomes of black cohosh (Actaea racemosa), an important botanical dietary supplement for women's health, and three closely related American Actaea species, A. rubra, A. pachypoda and A. podocarpa. The method was validated with respect to sensitivity, linearity, precision, accuracy and recovery. The total content of eight major polyphenols in the dried root and rhizome of the four species was determined to be from 0.36 to 2.92% (w/w). The antioxidant activities of Actaea extracts and polyphenolic compounds isolated from A. racemosa were evaluated on 1.1-diphenyl-2-picrylhydrazyl (DPPH) free radicals scavenging assay. The radical scavenging activity of the Actaea extracts correlates to their polyphenolic composition. This validated HPLC method can be used to distinguish A. racemosa from the other major American Actaea species based on this study.