The development of chemical probes for the investigation of the complex phosphorylation signaling cascades that regulate biological events is crucial to understanding these processes. We describe herein a bifunctional probe that enables spatial and temporal release of a biologically active ligand while allowing simultaneous monitoring of its binding to the protein of interest. Substitution of Tyr(-2) for the enviromentally sensitive fluorescent amino acid DANA in the sequence RLYRpSLPA which is known to bind the 14-3-3 protein does not adversely affect binding affinity and allows monitoring of the binding process. The binding of the peptide to 14-3-3 places the fluorescent reporter unit into a hydrophobic pocket, which changes the fluorescent maximum emission intensity and wavelength. At the same time, the newly developed photolabile 1-(2-nitrophenyl)ethyl-caged phosphoserine allows control of the release of the biologically active ligand through unmasking of the key phosphoserine functionality upon UV irradiation.