We describe an optimised protocol for application of isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass spectrometry to obtain relative quantitative data from peptides derived from tryptic digestions of proteins fractionated by using the 2D liquid-phase ProteomeLab PF 2D technique. This methodology is suitable for the quantitation of proteins from a pool of co-eluting proteins which are often difficult to identify for the purpose of candidate protein selection for biologically relevant qualitative/quantitative changes under experimental conditions or in disease states. iTRAQ quantitation also facilitates the possibility of result to result comparison using other methodologies such as UV protein quantitation via the ProteomeLab PF 2D technique. The optimised protocol outlined here allows relative quantitation by MALDI-TOF/TOF mass spectrometry with high sensitivity and without the need to perform 2D HPLC separation of labelled peptides. The overall outcome is the simplification in the data complexity and the ease of use of the labelling protocol.