Two-dimensional gel electrophoresis (2DE) is a proven high-resolution separation technology in the protein laboratory. Nevertheless, ever since its invention the technique has had the drawback of low reproducibility. For protein expression analysis based on replicates, differential gel electrophoresis (DIGE) has, therefore, been invented. This technique is however not applicable for the comparison of singular samples. With comparative fluorescence 2DE (CoFGE) a method was developed, which creates a protein reference net for gel matching on top of the gel-separated protein analyte. This internal standard enables both the correction of the protein spot coordinates and the estimation of the analyte concentration. It extends the use of 2DE and truly enables the use of searchable, browser-based 2DE databases. •Two-dimensional gel electrophoresis (2DE) is a major separation tool in the life sciences.•DIGE (differential gel electrophoresis) ensures reproducibility in protein expression analysis.•Comparative fluorescence 2DE (CoFGE) introduces standardisation by using a reference grid.•CoFGE generates reproducible protein spot coordinates in singular experiments supporting 2DE databases.•CoFGE allows quantification via the internal standard.