Purpose: The mechanism of axial elongation in the myopic eyeball remains to be elucidated. The retina is a delicate tissue that receives visual input and transmits the molecular signals to guide the eye growth. Reliable ocular biometric data have provided evidence to support the use of C57BL/6 mouse as the animal model susceptible to experimental myopia. We performed biometric measurements of the eyes in normal growing C57BL/6 mouse and used proteomics approach to establish a proteomic database of neural retina, revealing expressed proteins related to the developmental pathways and providing the basis for examining the molecular mechanisms underlying the development of myopia in future study. Methods:Three C57BL/6 mouse retina were obtained at postnatal day 46. Retinas were lysed with 5% SDS buffer. Proteins were tryptic digested with suspension traps (S-Traps). Peptides in each retina were separated into 6 fractions with high-pH peptide reversed-phase fractionation. In total, 18 fractions of peptides were analyzed with the data-dependent acquisition by using Sciex TripleTOF® 6600 mass spectrometer followed by bioinformatics analysis. Results: A total of 7122 non-redundant proteins (n=3, 1% FDR) were identified in the normal fractionated mouse retina. High-confidence proteins were identified in each fraction, with 909, 1780, 1674, 1876, 1620 and 1576 proteins respectively (n=3, 2 technical replicates). In result of 1612 fraction-specific proteins. KEGG pathway analysis revealed protome with high coincidence to reported signalling pathways(number of proteins), such as MAPK(91), ras(78), mTOR(64), axon guidance(64), cAMP(56), insulin(51); synapses, including glutamatergic(50), Dopaminergic(45), GABAergic(40) and cholinergic(40). Conclusion: Mass spectrometry enabled efficient screening of pathways involved in experimental condition. S-Trap protocol provides highly efficient and less complicated sample preparation for mouse retinal proteomics study. The comprehensive C57BL/6 mouse retinal proteome database with enriched pathways interested in myopia study, shall take advantage with the use of SWATH-MS acquisition to quantify multiple pathways in single experiment.