Two-dimensional difference gel electrophoresis (2D-DIGE) has been a staple of protein studies for almost three decades since first described in 1997. Although the advent of omic technologies has greatly expanded protein research and discovery, 2D-DIGE has consistently been the mainstay in biomedical applications. Differential protein expression is a hallmark of many disease states and identification of these biomarkers can improve diagnosis, prognosis and treatment. In this review, we examine the use of 2D-DIGE in exploring the cellular environment in physiologic and pathophysiologic states. We highlight this technology in protein identification and quantification, functional modification and biochemical pathways of interest. 2D-DIGE remains a useful tool due low cost and high resolving power for comparative and quantitative purposes in assessing disease states and facilitating identification of unique and novel biomarkers.