Human diseases like cancer, diabetes, and neurological disorders often arise from the dysfunction of multiple interconnected signaling pathways. To advance our ability to predict, diagnose, and treat these diseases, it is crucial to comprehend the intricate relationships between individual signaling components under varying cellular conditions. Molecular imaging tools, such as FRETbased biosensors, have been instrumental in visualizing dynamic cellular interactions in real-time within living cells. However, FRET has limitations, particularly its limited dynamic range. To address this, we propose a novel approach: the development of a suite of kinase activity reporters called ExRai-KARs (excitation ratiometric-based kinase activity reporters). Specifically, we are developing ExRai-based KARs for the Never-in-Mitosis A-related (NEK) and Ribosomal S6 (S6K) kinases. These biosensors offer the potential to characterize the dynamic signaling properties of NEK and S6K kinases with exceptional specificity and resolution within their native cellular environment. Our ExRai-KARs were designed based on a versatile molecular cloning framework derived from the existing PKA sensor, ExRai-AKAR2. We evaluated the dynamic characteristics of these biosensors using both in vitro biochemical techniques (fluorescent assay and ADP-Glo assay) and in situ live-cell imaging in mammalian HEK293T (NEK) and HeLa cell lines. Intriguingly, our ExRai-NEKAR biosensor exhibited a remarkable ~14-fold increase in its excitation ratio (395 nm/475 nm) when NEK1 and ATP were present in vitro. Notably, substituting ATP with the non-hydrolyzable ATP analog, AMP-PNP, resulted in no change in the excitation ratio, indicating that the observed alterations in ExRai-NEKAR's excitation ratio rely on NEK1- mediated phosphorylation rather than protein-protein interactions. Furthermore, while NEK5 efficiently phosphorylated ExRai-NEKAR, leading to a robust response, NEK2 or NEK9 incubation caused only marginal changes in the excitation ratio. This is consistent with previous 2 studies suggesting that NEK1 and NEK5 share a similar consensus motif distinct from those preferred by NEK2 and NEK9. Similarly, our ExRai-S6KAR biosensor exhibited a two-fold increase in its excitation ratio upon characterization in the presence of an active kinase. Live cell imaging confirmed a similar rise in fluorescent intensity upon activation of both NEK and S6K kinases, thus further validating our initial results. Based on our findings, we conclude that our biosensors are efficiently phosphorylated by their respective target kinases, making them valuable tools for monitoring real-time changes in the activity profiles of specific NEK family members and S6K kinase with high spatiotemporal resolution under diverse cellular conditions. This approach can provide insights into the regulation of complex signaling networks and potentially help identify biomarkers for associated diseases. Furthermore, these biosensors can be employed to investigate the response of these kinases to pharmacological, toxicological, and pathological agents, enhancing our understanding of disease mechanisms and aiding in the development of targeted therapies.