The 2DE is a powerful proteomic technique, with excellent protein separation capabilities where intact proteins are spatially separated by pI and molecular weight. 2DE is commonly used in conjunction with MS to identify proteins of interest. Current 2DE workflow requires several manual processing steps that can lead to experimental variability and sample loss. One such step is the transition between first dimension IEF and second-dimension SDS-PAGE, which requires exchanging denaturants and the reduction and alkylation of proteins. This in-solution-based equilibration step has been shown to be rather inefficient, losing up to 30% of the original starting material through diffusion effects. We have developed a refinement of this equilibration step using agarose stacking gels poured on top of the second-dimension SDS-PAGE gel, referred to as in-gel equilibration. We show that in-gel equilibration is effective at reduction and alkylation in SDS-PAGE gels. Quantification of whole-cell extracts separated on 2DE gels shows that in-gel equilibration increases protein retention, decreased intergel variability, and simplifies 2DE workflow.