Flow injection (FI)-CE coupled with frontal analysis (FA) was applied to the study of stereoselectivity binding of amlodipine (AL) to HSA. Under protein-drug binding equilibrium, the unbound concentrations of drug enantiomers were measured by plateau height. The stereoselectivity of AL binding to H SA was proved by the different free fractions of two enantiomers. In physiological phosphate solution (pH 7.4, ionic strength 0.17) when 200 mu M (+/-)AL was equilibrated with 300 mu M HSA, the concentration of unbound R-AL was about 1.5 times higher than that of its antipode. The binding constants of two enantiomers, KR-AL and K-S.AL, were 9910-11200 and 90200-104000 M-1, respectively. The results obtained by the method were compared with those determined by conventional equilibrium dialysis (ED)-CE and fluorescence spectra. Hydroxypropyl-beta-CD (HP-beta-CD) (10 mM) was used as a chiral selector in pH 3.7 phosphate buffer. L-Tryptophan (L-try) and ketoprofen (Ket) were used as displacement reagents to investigate the binding sites of AL to HSA. A binding synergism effect between hydrochlorothiazide (QL) and AL was observed and the results suggested that QL can destroy binding equilibrium of R-AL and S-AL toward HSA and they can occupy the same binding site of HSA (site 1). The reproducibility was confirmed by RSD (RSD<1.5%) of the plateau height determined by FI-CE frontal analysis (FI-CE-FA). The FI-CE-FA was a good method to study protein-drug interaction.