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 isotope-enriched medium or chemical or enzymatic modifications. Recently, an MS/MS-based quantitation method (iTRAQ) has been developed. This tutorial will cover overview of iTRAQ technology and the software for iTRAQ data analysis, ProteinPilot. Sample iTRAQ data, analysis and how to work with the results for each of the searches available in ProteinPilot software will be described. Some other computational tools to compare multiple iTRAQ experiments and the integration of protein annotations into a ProteinPilot protein summary will also be discussed.