Two-dimensional difference gel electrophoresis (DIGE) is a tool for measuring changes in protein expression between samples involving pre-electrophoretic labeling with cyanine dyes. Here we assess a common method to analyze DIGE data using the DeCyder software system. Experimental error was studied by a series of same sample comparisons. Aliquots of sample were labeled with N-hydroxyl succinimidyl ester-derivatives of Cy2, Cy3, and Cy5 dyes and run together on one gel. This allowed assessment of how experimental error influenced differential expression analysis. Bias in the log volume ratios was observed, which could be explained by differences in dye background. Further complications are caused by significant gel-to-gel variation in the spot volume ratio distributions. Using DeCyder alone results in an inability to define ratio thresholds for 90 or 95% confidence. An alternative normalization method was thus applied which resulted in improved data distribution and allowed greater sensitivity in analysis. When combined with a standardizing function, this allowed gel-independent thresholds for 90% confidence. The new approach, detailed here, represents a method to greatly improve the success of DIGE data analysis.