Systematic and rapid profiling of the interactions between small molecule drugs (or candidates) and target proteins is vital in studying drug promiscuity and drug discovery. However, a label-free and high-throughput method for the detection of small molecules binding on protein microarray is still lacking. In this research, we introduced surface plasmon resonance imaging (SPRi) technology combined with protein microarray on 3D-dextran hydrogel chip surface to realize the detection of small molecule drugs. With the development and optimization of this method, we obtained the ideal binding signals of small molecular drugs versus their target proteins, with good quality and uniformity (CV = 10.3%, n = 48). Then we demonstrated three applications, including in situ mimicking and detecting the formation of FK506-mediated trimer complex (FKBP12-FK506-calcineurin), rapid validation of FK506-FKBP12 recognition via FKBP12 mutant microarray (1 vs n mode), and 3 x 4 orthogonal studying the drug binding specificity on protein microarray (m vs n mode). Fortunately, these trials enable the label-free and kinetic study of drug-protein interactions on SPRi in a high-throughput manner, which could be a promising solution in screening/forecasting side effect of drugs (or candidates), drug repositioning or repurposing, target identification of natural products and personalized medicine, etc. (C) 2018 Published by Elsevier B.V.