The complexity and specificity of many forms of signal transduction are widely suspected to require spatial microcompartmentation and dynamic modulation of the activities of signaling molecules, such as protein kinases, phosphatases and second messengers. We have developed a series of fluorescent biosensors to probe the compartmentalized signaling activities. In this talk, I will focus on cAMP/PKA and PI3K/Akt/mTORC1 signaling pathways and present studies where we combined genetically encoded fluorescent biosensors, superresolution imaging, and targeted biochemical perturbations to probe the biochemical activity architecture of the cell.