Difference in-gel electrophoresis (DIGE) is a recent adaptation of conventional two-dimensional gel electrophoresis (2-DE) that incorporates novel fluorescent labels, has multiplex attributes, and boasts software-assisted image analysis. Combined, these characteristics offer significant benefits in accuracy and reproducibility to quantify differential protein expression levels between biological samples. The DIGE technique and materials required to perform it are described in detail within. The principles behind consistent gel image acquisition and reliable image analysis are also considered. Within the context of biomarker and drug target discovery, this method simplifies analysis, increases sample throughput, and represents a reliable 2-DE platform.