The determination of differences in relative protein abundance is a critical aspect of proteomics research that is increasingly employed to answer diverse biological questions. The Association of Biomolecular Resource Facilities (ABRF) Proteomics Research Group (PRG) 2006 study was a quantitative proteomics project in which the aim was to determine the identity and the relative amounts of eight proteins in two mixtures. There are numerous methodologies available to study the relative abundance of proteins between samples, but to date, there are few examples of studies that have compared these different approaches. For the 2006 PRG study, there were 52 participants who used a wide variety of gel electrophoresis-, HPLC- and mass spectrometry-based methods for relative quantitation. The quantitative data arising from this study were evaluated along with several other experimental details relevant to the methodologies employed.