In evaluating kinase inhibitors, kinetic parameters such as association/dissociation rate constants are valuable information, as are equilibrium parameters KD and IC50 values. Surface plasmon resonance (SPR) is a powerful technique to investigate these parameters. However, results are often complicated because of impaired conformations by inappropriate conditions required for protein immobilization and/or heterogeneity of the orientation of immobilization. In addition, conventional SPR experiments are generally time-consuming. Here we introduce the use of single-site specifically biotinylated kinases combined with a multichannel SPR device to improve such problems. Kinetic parameters of four compounds-staurosporine, dasatinib, sunitinib, and lapatinib-against six kinases were determined by the ProteOn XPR36 system. The very slow off-rate of lapatinib from the epidermal growth factor receptor and dasatinib from Bruton's tyrosine kinase and colony stimulating factor 1 receptor (CSF1R) were confirmed. Furthermore, IC50 values were determined by an activity-based assay. Evaluating both physicochemical and biochemical properties would help to understand the detailed character of the compound.