The complexity and specificity of cellular processes require spatial microcompartmentation and dynamic modulation of the underlying biochemical activities, such as dynamic phosphorylation and dephosphorylation catalyzed by specific protein kinases and phosphatases, respectively. We hypothesize that cellular biochemical activities are spatially organized into an “activity architecture” and reorganization and restructuring of this activity architecture lead to disease. In this talk, I will introduce a series of genetically encoded fluorescent biosensors that we have developed to monitor biochemical events in living cells, and then present a couple of studies where we combine quantitative fluorescence imaging with targeted perturbations as well as biochemical and functional assays to probe the subcellular regulation of cAMP/PKA and ERK signaling pathways.