Isobaric tag for relative and absolute quantitation (iTRAQ) is an in vitro labeling technique, where the peptides are labeled with specific tags, which is used for quantitation during MS analysis.  Since MS is capable of measuring the mass (or m/z) of the peptide, the tags are so designed that for each sample the tag remains unique and at the same time for all the samples pooled together, the overall mass of various tags remain the same. The iTRAQ reagent contains a reporter group, which is lost during fragmentation at MS/MS level. This reporter group is different for different iTRAQ tags and they differ from each other by mass of 1 Da. Thus, when the peptide identified by the first mass analyzer, is moved into the second mass analyzer for further fragmentation, the reporter ion is released and is measured by the detector. According to the number of samples, reporter ions are generated and hence their absolute quantitation is a measure of quantitation of peptide and hence protein in various samples. The advantage of iTRAQ is the ability to perform multiplexing, i.e. analyzing more than two samples at the same time. Currently, iTRAQ reagents are available with four or eight reporter groups and hence, at a time four (4-plex) or eight (8-plex) samples can be analyzed and quantified. Proteins from different samples are digested with trypsin, and peptides so obtained are differentially labeled with the iTRAQ reagents. The iTRAQ reagents interact with the primary amine or the N-terminal region of the protein and thus there is no biasness for any particular amino acid, which is a limitation in some quantitative proteomic techniques (Fig 1).