<p>G-protein-coupled receptors (GPCRs) control various downstream signaling pathways, with multiple effectors whose interactions are subject to sophisticated regulation to achieve signaling specificity. Spatiotemporal organization of GPCR signaling is essential for efficient control of multifaceted signaling pathways. To study how this spatiotemporal signaling is structured and affects cellular functionality, various genetically encoded molecular tools that can detect and perturb the target biochemical activities at a subcellular level have been developed. In this Review, we introduce various types of fluorescent protein-based biosensors and molecular tools that allow us to directly elucidate the spatiotemporal mechanisms of GPCR signaling regulation at a subcellular level. Finally, we highlight several applications of these molecular tools to study the spatiotemporal organization of GPCR in living cells to obtain a comprehensive understanding of the signaling architecture.</p>