The efficiency and reproducibility of two-dimensional difference gel electrophoresis (2D DIGE) depends on several crucial steps: (i) adequate number of replicate gels, (ii) accurate image acquisition, and (iii) statistically confident protein abundance analysis. The latter is inherently determined by the image analysis system. Available software solutions apply different strategies for consecutive image alignment and protein spot detection. While DeCyderTM performs spot detection on single gels prior to the alignment of spot maps, SameSpotsTM completes image alignment in advance of spot detection. In this study, the performances of DeCyderTM and SameSpotsTM were compared considering all protein spots detected in 2D DIGE resolved proteomes of three different environmental bacteria with minimal user interference. Proteome map-based analysis by SameSpotsTM allows for fast and reproducible abundance change determination, avoiding time-consuming, manual spot matching. The different raw spot volumes, determined by the two software solutions, did not affect calculated abundance changes. Due to a slight factorial difference, minor abundance changes were very similar, while larger differences in the case of major abundance changes did not impact biological interpretation in the studied cases. Overall, affordable fluorescent dyes in combination with fast CCD camera-based image acquisition and user-friendly image analysis still qualify 2D DIGE as a valuable tool for quantitative proteomics.