INTRODUCTIONA commercial reagent, known as the isobaric tag for relative and absolute quantification (iTRAQ), makes it possible to analyze multiple samples simultaneously. The ability of iTRAQ to compare relative protein abundances across as many as eight samples is a significant advantage over other stable isotope strategies, such as stable isotope labeling by amino acids in cell culture (SILAC) and isotope-coded affinity tag (ICAT). The iTRAQ protocol set compares the proteome of Saccharomyces cerevisiae under two different metabolic states (fermentation versus respiration) with the goal of determining the proteins involved in each of the activated pathways by measuring their relative abundances using iTRAQ. This protocol describes the preparation of yeast peptides in different metabolic states for iTRAQ analysis.