Abstract: 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 immobilized metal affinity chromatography (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. Stable isotope labeling of yeast proteomes was achieved by incorporation of arginine-13C6 and lysine-13C6 in a double auxotroph yeast strain. Pheromone treated yeast cells were mixed with isotope encoded cells as the control, 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. Out of more than 700 identified phosphopeptides, 139 were differentially regulated at least two-fold in response to mating pheromone. Among these regulated proteins were components belonging to the MAP kinase signaling pathway and to downstream processes including transcriptional regulation, the establishment of polarized growth and the regulation of the cell cycle.