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Background: Recently, it has become clear that drug-target residence time, in addition to affinity, often drives pharmacodynamic activity and disease efficacy in vivo. In the aim of determining the dissociation properties of test compounds, the kinase field has traditionally utilized purified proteins in various competitive probe displacement formats. Using such detection formats, kinetic parameters of drug-target binding can be quantified using analytical approaches described by Motulsky and Mahan. While quantitative, such approaches may fail to translate with cellular efficacy due to reliance on pure proteins or protein fragments. Furthermore, these methods are generally not amenable to high-throughput analysis for exploring intracellular drug-target residence time. Thus, a simple and quantitative method to evaluate the residence time of compounds inside living cells is highly desirable. Method: The NanoBRET™ Target Engagement Intracellular Kinase Assay was employed to investigate the kinetics of target engagement for various test compounds in living cells. This method utilizes BRET (Bioluminescence Resonance Energy Transfer) in cells by molecular proximity of the NanoBRET™ Tracer to the NanoLuc® luciferase-fused kinase. Quantitative determination of both of apparent affinity as well as binding kinetics for unodified test compounds can be achieved by competitive displacement of the Tracer. Full-length BTK fused with the NanoLuc® luciferase was transiently transfected in HEK293 cells and the cells were incubated at 37°C, 5% CO2 overnight. Transfected cells were harvested and suspended in 1 ml of Assay Medium in a conical tube. After incubation with test compound for 2 hours, the cells were washed with Assay Medium to remove unbound compound. Dosing of test compounds was an approximately IC80-90 concentration unless otherwise specified. The cell suspension was dispensed into a 96-well plate and the NanoBRET™ Tracer K-4 or K-5 was added to the wells following the addition of the Nano-Glo® Substrate Solution. BRET was measured repeatedly with the Glomax® Discover Multimode Microplate Reader. Data were fitted to the one-phase association equation or the kinetic equation developed by Malany to obtain the dissociation rate constatns of test compounds. Results: The kinetic constants using tracer K-4 or K-5 were successfully quantitated in live cells using NanoBRET™. Though the kinetic constants as determined by simple kobs fitting using tracer K-4 or K-5 were different, the constants as determined using the Malany approach for each tracer were in gratifying agreement. The results determined using the Malany equation reveal suitable differentiation of residence time for the irreversible and reversible compounds. As expected, longer residence time was observed for the irreversible/covalent inhibitors. Some reversible inhibitors also showed protracted residence time, offering opportunities for durable target inhibition in cells. Conclusion: We propose the method by which the data in the compound wash-out experiments are fitted to the kinetic equation of Malany as a simple and quantitative method to determine intracellular residence time of kinase inhibitors. Citation Format: Takeomi Inoue, Aki Emi, James D Vasta, Matthew B Robers, Yusuke Kawase. A new method to determine drug-target residence time of kinase inhibitors in living cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C085. doi:10.1158/1535-7163.TARG-19-C085