Two‐dimensional gel electrophoresis (2‐DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteomics. In spite of promising alternative or complementary technologies (e.g. multidimensional protein identification technology, stable isotope labelling, protein or antibody arrays) that have emerged recently, 2‐DE is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. 2‐DE enables the separaration of complex mixtures of proteins according to isoelectric point (pI), molecular mass (M_r), solubility, and relative abundance. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or post‐translational modifications. This is in contrast to liquid chromatography‐tandem mass spectrometry based methods, which perform analysis on peptides, where M_r and pI information is lost, and where stable isotope labelling is required for quantitative analysis. Today's 2‐DE technology with IPGs (Görg et al., Electrophoresis 2000, 21, 1037–1053), has overcome the former limitations of carrier ampholyte based 2‐DE (O'Farrell, J. Biol. Chem. 1975, 250, 4007–4021) with respect to reproducibility, handling, resolution, and separation of very acidic and/or basic proteins. The development of IPGs between pH 2.5–12 has enabled the analysis of very alkaline proteins and the construction of the corresponding databases. Narrow‐overlapping IPGs provide increased resolution (δpI = 0.001) and, in combination with prefractionation methods, the detection of low abundance proteins. Depending on the gel size and pH gradient used, 2‐DE can resolve more than 5000 proteins simultaneously (˜2000 proteins routinely), and detect and quantify < 1 ng of protein per spot. In this article we describe the current 2‐DE/MS workflow including the following topics: sample preparation, protein solubilization, and prefractionation; protein separation by 2‐DE with IPGs; protein detection and quantitation; computer assisted analysis of 2‐DE patterns; protein identification and characterization by MS; two‐dimensional protein databases.