The retina is a key sensory tissue composed of multiple layers of cell populations that work coherently to process and decode visual information. Mass spectrometry-based proteomics approach has allowed high-throughput, untargeted protein identification, demonstrating the presence of these proteins in the retina and their involvement in biological signalling cascades. The comprehensive wild-type mouse retina proteome was prepared using a novel sample preparation approach, the suspension trapping (S-Trap) filter, and further fractionated with high-pH reversed phase chromatography involving a total of 28 injections. This data-dependent acquisition (DDA) approach using a Sciex TripleTOF 6600 mass spectrometer identified a total of 7,122 unique proteins (1% FDR), and generated a spectral library of 5,950 proteins in the normal C57BL/6 mouse retina. Data-independent acquisition (DIA) approach relies on a large and high-quality spectral library to analyse chromatograms, this spectral library would enable access to SWATH-MS acquisition to provide unbiased, multiplexed, and quantification of proteins in the mouse retina, acting as the most extensive reference library to investigate retinal diseases using the C57BL/6 mouse model.