The sclera is white, tough outer covering, known for providing structural support to the eyeball and the intraocular contents. Any abnormality in sclera results in disorders such as conjunctivitis, episcleritis, scleritis and hence a proper investigation will help in preventing the same. In the current study, we carried out an in-depth proteomic analysis of human sclera in which the proteins were extracted from the sclera tissue and subjected to both in-gel and in-solution digestion using trypsin as the proteolytic enzyme. The peptides were analyzed using Orbitrap Fusion Tribrid mass spectrometerand the data acquired was searched against Human RefSeq 75 protein database using SEQUEST HT and Mascot which resulted in the identification of 4,493 non-redundant proteins.The outcome of this study will facilitate in discovery of potential candidate biomarkers aiding in curbing the scleral disorders.