The method uses high-speed countercurrent chromatography to retain a given concentration of the chiral selector (CS) in the liquid stationary phase. A minute amount of an enantiomeric analyte is eluted through the column to measure its retention time from which the distribution ratio of the analyte is computed. The experiment is repeated by varying the CS concentration in the stationary phase. Using a set of data thus obtained, plotting the CS concentration in the stationary phase against the relative distribution ratio of the analyte produces a straight line whose slope corresponds to the formation constant (Kf) of the CS-analyte complex. The validity of the method is demonstrated on a set of dinitrobenzoyl amino acids using N-dodecanoyl–L-proline-3,5-dimethylanilide as CS. The method will be useful for understanding the basic mechanism of enantioselectivity and designing effective chiral selectors.