The technique of fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) for differential protein expression analysis has been evaluated using a model breast cancer cell system of ErbB-2 overexpression. Labeling of paired cell lysate samples with NHS ester-derivatives of fluorescent Cy3 and Cy5 dyes for separation on the same 2D gel enabled quantitative, sensitive and reproducible differential expression analysis of the cell lines. SyproRuby staining was shown to be a highly sensitive and 2D-DIGE-compatible method for post-electrophoretic visualization of proteins, which could then be picked and identified by MALDI mass spectroscopy. Indeed, from these experiments, we have identified multiple proteins that are likely to be involved in ErbB-2-mediated transformation. A triple dye labeling methodology was used to identify proteins differentially expressed in the cell system over a time course of growth factor stimulation. A Cy2-labeled pool of samples was used as a standard with all Cy3- and Cy5-labeled sample pairs to facilitate cross-gel quantitative analysis. DeCyder (APBiotech) software was used to distinguish clear statistical differences in protein expression over time and between the cell lines.