An RP-HPLC method for the determination of daidzein, genistein, formononetin and biochanin A in red clover (Trifolium pratense L.) was developed and validated. The compounds are quantified after hydrolytic extraction using an internal standard. On a base-deactivated C(18) column good separation of the analytes, also from accompanying substances, and excellent peak shape are achieved by gradient elution with aqueous sulfuric acid and acetonitrile. The method was applied to the analysis of different red clover cultivars.