The aqueous humor is a colourless, transparent fluid that fills the anterior chamber of the eye. It has an important role to play in maintaining the intraocular pressure of the eye and providing nourishment to avascular structures such as the lens and cornea. It is also essential for providing a clear path for the entry and exit of light from the eye and maintaining the refractive power of the eye. In this study, 250 samples of aqueous humor were collected from patients undergoing cataract surgery. The samples were pooled, divided into aliquots and subjected to ingel digestion, insolution digestion followed by strong cation exchange chromatography and basic reverse phase liquid chromatography fractionation. MS/MS analysis was carried out on a Fourier transform LTQ-Orbitrap velos mass spectrometer and the data were searched with the search algorithms Mascot, SEQUEST and X!Tandem against the NCBI RefSeq human protein database. We have identified 818 proteins of which 447 have been identified for the first time. Proteins such as sorbitol dehydrogenase, filensin and phakinin were some of the novel proteins identified in this study. Classification of proteins based on domain information yielded 304 proteins with signal peptides, 46 proteins with transmembrane domain and 75 proteins with both signal peptide and transmembrane domains. Our study provides a detailed profile of the human aqueous humor. The results from this study of aqueous proteins should help facilitate research into pathological conditions of the eye such as glaucoma, myopia, cataract and uveitis.