One of the hallmarks of blood bank stored red blood cells (RBCs) is the irreversible transition from a discoid to a spherocyte‐like morphology with membrane perturbation and cytoskeleton disorders. Therefore, identification of the storage‐associated modifications in the protein–protein interactions between the cytoskeleton and the lipid bilayer may contribute to enlighten the molecular mechanisms involved in the alterations of mechanical properties of stored RBCs. Here we report the results obtained analyzing RBCs after 0, 21 and 35 days of storage under standard blood banking conditions by label free mass spectrometry (MS)‐based experiments. We could quantitatively measure changes in the phosphorylation level of crucial phosphopeptides belonging to β‐spectrin, ankyrin‐1, α‐adducin, dematin, glycophorin A and glycophorin C proteins. Data have been validated by both western blotting and pseudo …