Two‐dimensional difference gel electrophoresis (DIGE) is a tool for measuring changes in protein expression between samples involving pre‐electrophoretic labeling with cyanine dyes. In multi‐gel experiments, univariate statistical tests have been used to identify differential expression between sample types by looking for significant changes in spot volume. Multivariate statistical tests, which look for correlated changes between sample types, provide an alternate approach for identifying spots with differential expression. Partial least squares‐discriminant analysis (PLS‐DA), a multivariate statistical approach, was combined with an iterative threshold process to identify which protein spots had the greatest contribution to the model, and compared to univariate tests for three datasets. This included one dataset where no biological difference was expected. The novel multivariate approach, detailed here, represents a method to complement the univariate approach in identification of differentially expressed protein spots. This new approach has the advantages of reduced risk of false‐positives and the identification of spots that are significantly altered in terms of correlated expression rather than absolute expression values.