GAPDH, β-actin, and β-tubulin are essential housekeeping proteins commonly used as reference controls for protein expression studies. GAPDH is a key glycolytic enzyme that facilitates the production of cellular energy, while β-actin and β-tubulin are major structural components of the cytoskeleton. Besides their well-established housekeeping functions, emerging studies have demonstrated critical roles for these proteins in brain developmental and pathological processes. However, few studies have examined how the expression patterns of these proteins change throughout mammalian brain development to adulthood. Considering the dynamic structural and functional changes that occur during brain development and the roles of GAPDH, β-actin, and β-tubulin in related biological processes, we investigated the developmental expression levels of these proteins in the mouse cortex at various embryonic (E15-P0) and postnatal (P0-P20, adult) stages using western blotting analysis with total protein normalization. We identified a substantial increase in GAPDH protein levels and a decrease in β-actin and β-tubulin in protein levels in the mouse cortex between birth and early adulthood, which occurred during the second week of postnatal life. Analysis of RNA-seq data from the ENCODE Consortium revealed correlated changes at the RNA transcript level. Overall, our study reveals robust age-dependent changes in cortical GAPDH, β-actin, and β-tubulin expression levels during mouse postnatal development and suggests precautions when using these proteins as reference controls in cortical development studies.