Gallus gallus avian eggshell is composed of 95% calcium carbonate on calcitic form and of 3.5% extracellular organic matrix (proteins, polysaccharides and proteoglycans). This highly ordered structure with a polycrystalline organization result of the control of mineral deposition by the organic matrix components in the lumen of the uterus. This interaction leads to the eggshell ultrastructure and consequently contributes to its resulting mechanical properties. This study used GeLC MS/MS analyses combined to label free quantitative analysis to identify and quantify matrix proteins at the pivotal step of the calcification process (amorphous calcium carbonate deposit, amorphous calcium carbonate transformation into calcite, large calcite crystal units and rapid growth phase). The study gave new insight on proteins playing crucial role in the biomineralization of the shell.