Cellular processes such as proliferation, differentiation, and adaptation to environmental changes are regulated by protein phosphorylation. Development of sensitive and comprehensive analytical methods for determination of protein phosphorylation is therefore a necessity in the pursuit of a detailed molecular view of complex biological processes. We present a quantitative modification-specific proteomic approach that combines stable isotope labeling by amino acids in cell culture (SILAC) for quantitation with IMAC for phosphopeptide enrichment and three stages of mass spectrometry (MS/MS/MS) for identification. This integrated phosphoproteomic technology identified and quantified phosphorylation in key regulator and effector proteins of a prototypical G-protein-coupled receptor signaling pathway, the yeast pheromone response. SILAC encoding of yeast proteomes was achieved by incorporation of [(13)C(6)]arginine and [(13)C(6)]lysine in a double auxotroph yeast strain. Pheromone-treated yeast cells were mixed with SILAC-encoded cells as the control and lysed, and extracted proteins were digested with trypsin. Phosphopeptides were enriched by a combination of strong cation exchange chromatography and IMAC. Phosphopeptide fractions were analyzed by LC-MS using a linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. MS/MS and neutral loss-directed MS/MS/MS analysis allowed detection and sequencing of phosphopeptides with exceptional accuracy and specificity. Of more than 700 identified phosphopeptides, 139 were differentially regulated at least 2-fold in response to mating pheromone. Among these regulated proteins were components belonging to the mitogen-activated protein kinase signaling pathway and to downstream processes including transcriptional regulation, the establishment of polarized growth, and the regulation of the cell cycle.