Kinases are involved in numerous cellular processes but possibly also in tumor progression. Several kinase inhibitors are approved as drugs and there is an intense search for new inhibitors in pharmaceutical research. In this study, we present a new analytical method based on reflectometric interference spectroscopy, RIfS, for kinase and inhibitor screening. First, the sensor surface was optimized to reduce non-specific binding. Different inhibitors, e.g. staurosporine or fasudil, were immobilized on the transducer surface. Different kinases (focal adhesion kinase and cAMP-dependent protein kinase) were flushed over the sensor with the immobilized inhibitors. The specific interaction was proven by binding inhibition assays. The kinase-inhibitor interaction was monitored label-free and recorded in real time allowing the binding curves to be used to determine the association and dissociation rate constants as well as the affinity. These constants differed depending on the specific kinase-inhibitor pair, which was well expected from parallel docking simulations and measurements with microscale thermophoresis. The strategy was successfully transferred to 1-lambda reflectometry, a modification of RIfS, to enable the simultaneous monitoring of several kinase-inhibitor interactions in 5×7 small spots increasing throughput and automation on a sensor array with imaging detection. Importantly, the techniques developed here can provide both kinetic and thermodynamic data for a multitude of kinases in a single screening approach, which allows for both protein kinase and inhibitor screening.