Using classical genetics to study modular phosphopeptide-binding domains within a family of proteins that are functionally redundant is difficult when other members of the domain family compensate for the product of the knocked-out gene. Here we describe a chemical genetics approach that overcomes this limitation by using UV-activatable caged phosphopeptides. By incorporating a caged phosphoserine residue within a consensus motif, these reagents simultaneously and synchronously inactivate all phosphoserine/phosphothreonine-binding domain family members in a rapid and temporally regulated manner. We applied this approach to study the global function of 14-3-3 proteins in cell cycle control. Activation of the caged phosphopeptides by UV irradiation displaced endogenous proteins from 14-3-3-binding, causing premature cell cycle entry, release of G1 cells from interphase arrest and loss of the S-phase checkpoint after DNA damage, accompanied by high levels of cell death. This class of reagents will greatly facilitate molecular dissection of kinase-dependent signaling pathways when applied to other phosphopeptide-binding domains including SH2, Polo-box and tandem BRCT domains.