Cellular signaling networks are the foundation which determines the fate and function of cells as they respond to various cues and stimuli. The discovery of fluorescent proteins over 25 years ago enabled the development of a diverse array of genetically encodable fluorescent biosensors that are capable of measuring the spatiotemporal dynamics of signal transduction pathways in live cells. In an effort to encapsulate the breadth over which fluorescent biosensors have expanded, we endeavored to assemble a comprehensive list of published engineered biosensors, and we discuss many of the molecular designs utilized in their development. Then, we review how the high temporal and spatial resolution afforded by fluorescent biosensors has aided our understanding of the spatiotemporal regulation of signaling networks at the cellular and subcellular level. Finally, we highlight some emerging areas of research in both biosensor design and applications that are on the forefront of biosensor development.