New HPLC-chemometric approaches were proposed for the simultaneous chromatographic quantification of daidzein, genistein, formononetin, and biochanin A in the samples consisting of the aerial parts of Trifolium lucanicum Gasp. (Leguminosae). Partial least squares and principal component regression algorithms were applied to the multiple chromatographic data set obtained by measuring at 240, 248, 256, and 264 nm to construct HPLC-partial least squares and HPLC-principal component regression calibrations. Chromatographic separation was carried out by using a mobile phase containing methanol, acetate buffer (pH = 4.75) and acetonitrile (21:58:21, v/v/v) on the reversed phase column, Supelcosil (TM) LC-18 (15 cm x 4.6 mm id). In addition, conventional HPLC based on the detection at a single wavelength was used for the determination of each compound in the extracts of T lucanicum. The validity and applicability of the proposed HPLC-chemometric and conventional HPLC methods were performed by analyzing various synthetic plant samples. A good agreement was observed in the application of the proposed HP LC-chemometric tools to the synthetic and extracted samples of T lucanicum.