High-performance frontal analysis (HPFA) was used for a protein binding study of isoflavones (daidzein, genistin, and genistein), enantiomers of perillyl alcohol and S-ibuprofen to human serum albumin (HSA). The analyses were performed on a Develosil and Inertsil 100-Diol-5 column (10 cm×4.6mm). Sodium phosphate solution (pH 7.4, ionic strength 0.17) was used as the mobile phase at a flow rate of 1 ml/min. To ensure the drug to be eluted as a trapezoidal peak with a plateau, injection volumes were each fixed up the zonal profile with an evident plateau appears. The unbound drug concentration was determined from a plateau height of the plateau region after that experimental data were fitted by Scatchard equation. The binding constants (K) and total binding affinities (nK) of drugs to HSA were calculated, respectively.