Abstract Background Two-dimensional gel electrophoresis (2DE) is a powerful technique for studying protein isoforms and their modifications. Existing commercial 2D image analysis tools rely on spot detection that limits analysis of complex protein profiles, e.g. spot appearance/disappearance or overlapping spots. Pixel-by-pixel correlation analysis, an analysis technique for identifying relations between protein patterns in gel images and external variables, can overcome such limitations in spot analysis. Results We have implemented the first publically available pixel-by-pixel correlation analysis tool, the software Gel2DE. 2D immunoblot time course analysis of p53 protein stabilization in response to ionizing irradiation shows that pixel-by-pixel analysis can yield an overall activation biosignature for p53, despite changing spots shape, size and position. Conclusions Pixel-by-pixel correlation of aligned 2D images permits analysis of complex protein patterns. We anticipate that the Gel2DE correlation software will be a useful tool for future bioinformatics discoveries through 2D gel electrophoresis.