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Understanding the development of the malaria parasite within the mosquito vector at the molecular level should provide novel targets for interrupting parasitic life cycle and subsequent transmission. Availability of the complete genomic sequence of the major African malaria vector, Anopheles gambiae, allows discovery of such targets through experimental as well as computational methods. In the female mosquito, the salivary gland tissue plays an important role in the maturation of the infective form of the malaria parasite. Therefore, we carried out a proteomic analysis of salivary glands from female An. gambiae mosquitoes. Salivary gland extracts were digested with trypsin using two complementary approaches and analyzed by LC-MS/MS. This led to identification of 69 unique proteins, 57 of which were novel. We carried out a functional annotation of all proteins identified in this study through a detailed bioinformatics analysis. Even though a number of cDNA and Edman degradation-based approaches to catalog transcripts and proteins from salivary glands of mosquitoes have been published previously, this is the first report describing the application of MS for characterization of the salivary gland proteome. Our approach should prove valuable for characterizing proteomes of parasites and vectors with sequenced genomes as well as those whose genomes are yet to be fully sequenced.