AbstractDIGE is a protein labelling and separation technique allowing quantitative proteomics of two or more samples by optical fluorescence detection of differentially labelled proteins that are electrophoretically separated on the same gel. DIGE is an alternative to quantitation by MS‐based methodologies and can circumvent their analytical limitations in areas such as intact protein analysis, (linear) detection over a wide range of protein abundances and, theoretically, applications where extreme sensitivity is needed. Thus, in quantitative proteomics DIGE is usually complementary to MS‐based quantitation and has some distinct advantages. This review describes the basics of DIGE and its unique properties and compares it to MS‐based methods in quantitative protein expression analysis.