Two-dimensional gel electrophoresis (2DGE) continues to be a useful approach to study protein expression. Although liquid chromatographic and mass spectrometric approaches that overcome some of the limitations and labour intensity of 2DGE are increasingly popular, this electrophoretic approach still has exceptional relevance in toxicology. Despite the technical challenges, pharmacologists/toxicologists continue to use gel-based proteomics to assess the biological and health effects of chemical treatment and exposure. This brief review addresses the use of 2DGE-based proteomics in drug development and toxicology, emphasising its unique strengths and weaknesses, and considers recent developments in this strategy that have evolved to directly confront the issues of dynamic range and reproducibility that have previously limited the overall use of 2D electrophoresis.