Abstract We present a biophysical method to directly measure target engagement within intact mammalian cells using bioluminescence energy transfer (BRET). Compound interactions with intracellular targets can be detected with complete specificity by their ability to compete with energy transfer complexes introduced into the cells. These complexes can be detected at physiologically relevant levels by exploiting an extraordinarily bright luciferase (NanoLuc), together with fluorescent tracers optimized for cell-permeability and spectral resolution from the luciferase. We demonstrate applications of the technology for target engagement among key drug target classes, including; kinases, histone deacetylases (HDACs), bromodomains, and the methyltransferase EZH2. Intracellular selectivity and affinity profiles of various reference compounds and approved drugs will be presented. For a panel of HDAC inhibitors, affinity profiles for specific HDAC isozymes strongly correlate with phenotypic potencies (e.g. cell viability). Furthermore, the luminescent output of the energy transfer complex enables a technique to monitor ligand occupancy in real-time. Association and dissociation rates can be derived from the kinetic measurements, providing a means to quantify drug residence time on select targets within intact cell populations. This novel application of intracellular BRET should significantly advance target engagement work flows, and allow for intracellular target affinities to be coupled to phenotypic outcomes. Citation Format: Matthew B. Robers, Melanie Dart, Chad Zimprich, Thomas Kirkland, Sergiy Levin, Thomas Machleidt, Jim Hartnett, Kris Zimmerman, Rachel Ohana, Danette Daniels, Mei Cong, Frank Fan, Keith Wood. Measuring intracellular target engagement and drug residence time with nanoBRET. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3512. doi:10.1158/1538-7445.AM2015-3512