Proteomics research includes the characterization of protein mixtures in order to understand complex biological systems and determine relationships between proteins, their function, and protein-protein interactions. Established methods for relative quantitation of proteins involve growth on an isotope-enriched medium or chemical or enzymatic modifications. Recently, an MS/MS-based quantitation method (iTRAQ) has been developed. This tutorial will provide an overview of iTRAQ technology and ProteinPilot, the software for iTRAQ data analysis. Sample iTRAQ data, analysis, and how to work with the results for each of the searches available in ProteinPilot will be described. We will also discuss computation tools to compare multiple iTRAQ experiments and integration of protein annotations into the ProteinPilot protein summary.