Phosphorylation of proteins is involved in cell functions, of which cytoskeleton organization. In particular, the phosphotyrosine (pY) has been reported to play a role in red blood cell (RBC) functions. During the storage of RBC in the context of transfusion medicine, cells suffer from storage lesions that affect energy metabolism as well as cell morphology. In the present research work, RBCs were treated (in absence of calcium) with a protein tyrosine phosphatase inhibitor (orthovanadate, OV) to trigger phosphorylation allowing to investigate this effect on RBCs during the storage. Erythrocytes were studied by phosphoproteomics, Western blot analyses and cell morphology by digital holographic microscopy in presence of kinase inhibitors. The OV treatment triggered phosphorylation events, that disappeared during the storage. As a result, 609 phosphoproteins containing 5’298 phosphosites were detected in the membrane extract, of which 43 pY were up- or down-regulated. Following these phosphorylation processes, shape of RBCs shifted from discocytes to spherocytes, and the addition of kinase inhibitors (KIs) inhibited this transition by counteracting the OV treatment. In addition, the different KIs used highlighted potentially two mechanisms involving membrane proteins and playing a role in RBC shape. The capacity of RBCs to maintain their function is central in transfusion medicine and the presented results contribute to a better understanding of RBC biology.