Despite their fundamental importance, the dynamics of signaling pathways in living cells remain challenging to study, due to a lack of non-invasive tools for temporal assessment of signal transduction in desired cell models. Here we report a dual-reporter strategy that enables researchers to monitor signal transduction in mammalian cells in real-time, both temporally and quantitatively. This is achieved by co-expressing green fluorescent protein and firefly luciferase in response to signaling stimuli. To display the versatility of this approach, we constructed and assessed eight unique signaling pathway reporters. We further validated the system by establishing stable NF-kappa B pathway reporter cell lines. Using these stable cell lines, we monitored the activity of NF-kappa B-mediated inflammatory pathway in real-time, both visually and quantitatively. Live visualization has the power to reveal individual cell responses and is compatible with single cell analysis, In addition, we provide evidence that this system is readily amenable to a high-throughput format. Together, our findings demonstrate the potential of the dual reporter system, which significantly improves the capacity to study signal transduction pathways in mammalian cells. (C) 2014 Elsevier Inc. All rights reserved.