Protein phosphorylation is one of the most common forms of post-translational modification and is involved in the regulation of key signaling pathways in the cell. Dysfunctional phosphorylation plays a key role in various diseases, especially cancer and neurodegenerative disorders. Protein kinases have been the focus of intense recent interest by the pharmaceutical industry. Indeed, most new cancer drugs approved by the FDA in the last several years target kinases, and there are hundreds of new kinase inhibitors under development. To this end, it is very important to have quantitative methods for measuring changes in kinase activities. In vitro kinase activity assays take the target molecules out of cellular contexts. Fluorescent protein-based kinase biosensors have enabled the real-time monitoring of kinase activities within the native context of living cells, yet most commonly used biosensors exhibit poor sensitivity (e.g., dynamic range) for imaging physiological signaling activities in situ.