The recent trend in science is to assay as many biological molecules as possible within a single experiment. This trend is evident in proteomics where the aim is to characterize thousands of proteins within cells, tissues, and organisms. While advances in mass spectrometry have been critical, developments made in two-dimensional PAGE (2D-PAGE) have also played a major role in enabling proteomics. In this review, we discuss and highlight the advances made in 2D-PAGE over the past 25 years that have made it a foundational tool in proteomic research.