Two-dimensional electrophoresis has, for many years, been the primary workhorse for performing functional proteomics, the large-scale analysis of protein expression differences. Despite its merits, limitations inherent to this technology have been recognized for a long time, ranging from its gel-to-gel variability to its inability to represent several classes of proteins. Recently, however, technical advances in two-dimensional electrophoresis have alleviated several of these drawbacks. Fractionation approaches prior to two-dimensional electrophoresis, e.g. by chromatography, organelle fractionation or Equalizer Beads technology, have increased the number of visible proteins. Fluorescent two-dimensional difference gel electrophoresis has boosted the quantitative aspects of two-dimensional electrophoresis. New protein stains have also enabled the analysis of post-translationally modified proteins. As a result, two-dimensional electrophoresis has been thoroughly modernized, enabling it to remain the preferred method for protein expression analysis in a large number of laboratories. In this review we will give an overview of these technological advances.