Two‐dimensional difference gel electrophoresis (2‐D DIGE), in combination with mass spectrometry, is a highly effective method for the rapid and reproducible detection of differentially expressed proteins. This approach, however, has the unfortunate drawback that it preferentially displays rather abundantly expressed proteins. Nevertheless, comparison of the protein expression levels of the striate cortex of adult cats and 30‐day‐old kittens, resulted in the identification of several proteins related to postnatal brain development and possibly age‐dependent plasticity as well (Van den Bergh et al., J. Neurochem. 2003, in press). The goal of the present study was the selective enrichment and identification of less abundant proteins within the same paradigm. Hereto, we performed a reversed‐phase chromatography prefractionation of our tissue lysate to separate the proteins in four fractions based on their hydrophobicity prior to 2‐D DIGE analysis. This approach not only confirmed the differential expression levels of a number of proteins from the previous study, but also identified three additional proteins preferentially expressed in kitten visual cortex and five additional proteins with higher expression levels in adult cat visual cortex. These spots were not visible on the total tissue lysate protein maps, indicating that the high‐performance liquid chromatography (HPLC) prefractionation enabled us to visualize additional, less abundant proteins.