AbstractDrug–protein interaction analysis has become a considerable topic in life science which includes clarifying protein functions, explaining drug action mechanisms and uncovering novel drug candidates. This work was to determine the association constants (KA) of six drugs to β2‐adrenergic receptor by injection amount‐dependent method using stationary phase containing the immobilized receptor. The values of KA were calculated to be (25.85 ± 0.035) × 104 m−1 for clorprenaline, (42.51 ± 0.054) × 104 m−1 for clenbuterol, (6.67 ± 0.008) × 104 m−1 for terbutaline, (33.99 ± 0.025) × 104 m−1 for tulobuterol, (7.59 ± 0.011) × 104 m−1 for salbutamol and (78.52 ± 0.087) × 104 m−1 for bambuterol. This rank order agreed well with the data determined by zonal elution, frontal analysis and nonlinear chromatography, even using different batches of β2‐AR column. A good correlation was found between the association constants by the current method and radio‐ligand binding assay. Our data indicates that the injection amount‐dependent method is a powerful alternative for rapid analysis of ligand–receptor interactions.