The comparison of two-dimensional (2-D) gel images from different samples is an established method used to study differences in protein expression. Conventional methods rely on comparing images from at least 2 different gels. Due to the high variation between gels, detection and quantification of protein differences can be problematic. Two-dimensional difference gel electrophoresis (Ettan™ DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. In the application of DIGE different samples are labelled with mass and charge matched spectrally resolvable fluorescent dyes and are then separated on the same 2-D gel. Using an Escherichia coli lysate “spiked” with varying amounts of four different known proteins, we have tested a novel experimental design that exploits the sample multiplexing capabilities of DIGE, by including a standard sample in each gel. The standard sample comprises equal amounts of each sample to be compared and was found to improve the accuracy of protein quantification between samples from different gels allowing accurate detection of small differences in protein levels between samples.