Assays that furnish a fluorescent readout of protein kinase activity provide a means to identify and characterize inhibitory agents, assess structure-function relationships, and correlate enzyme activity with cellular behavior. Although several protein kinase sensors have been described in the literature, their fluorescent response to phosphorylation are generally modest to moderate (1.1 – 8-fold). We have developed a “deep quench” strategy that elicits a dramatic amplification of fluorescence (>60-fold) in cAMP-dependent protein kinase substrates. We describe sensor design, assay development via a combination of library synthesis and screening, characterization of the assay components, and an assessment of two inhibitory species.