Abstract Protein:protein interactions (PPIs) are essential to the cellular signal transduction pathways that contribute to cancer. Although numerous approaches exist to monitor protein:protein interactions in vitro, methods for intracellular detection have been more limited. We developed a system for the detection of PPIs based on NanoLuc luciferase. Proteins are fused to either an 18 kDa or 1 kDa subunit. PPI facilitates interaction of the subunits, leading to increased luminescence. Intracellular PPIs are measured using a non-lytic assay protocol by providing a cell permeable substrate. The interaction status can be monitored at a single time point or followed continuously over 1-2 hours. Unlike related approaches where a luciferase enzyme is simply split, the subunits of this system were optimized for structural stability and minimal binding affinity. The result is a luminescence signal that is orders of magnitude brighter than approaches using split luciferase, allowing use of fusion protein expression levels at or near physiological levels. The low affinity interaction between subunits (Kd ∼200 μM) minimizes assay background by preventing non-specific association, and the system shows rapid reversibility using model systems such as protein kinase A catalytic and regulatory subunits. We have successfully applied this system to several PPIs related to cancer research, including TP53:MDM2, MYC:MAX, and members of the RAS-RAF signaling pathway. Citation Format: Brock Binkowski, Andrew S. Dixon, Marie K. Schwinn, Mary P. Hall, Kris Zimmerman, Paul Otto, Thomas Lubben, Braeden Butler, Thomas Kirkland, Monika G. Wood, Lance P. Encell, Frank Fan, Keith V. Wood. A novel luminescent system for monitoring intracellular protein:protein interactions. [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 1998. doi:10.1158/1538-7445.AM2015-1998