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Sand fly saliva is known to play an important role in the establishment of Leishmania spp. infection. As a consequence, identifying antigenic salivary proteins of different leishmaniasis vectors has currently become a major task in the field of anti-Leishmania vaccine development. The purpose of this work was to improve the knowledge of Phlebotomus perniciosus salivary proteins by combining two-dimensional gel electrophoresis (2DE) methodology, mass spectrometry and Western blotting (WB). Salivary protein profiles of three P. perniciosus colonies from different geographic origins in Spain were compared through SDS-PAGE, leading to a similar pattern with no qualitatively noticeable differences. A gradual increase of the protein content was significantly detected with the age of sand flies, reaching the complete salivary protein profiles at day four. The 2DE revealed a reproducible protein profile that matched the classic monodimensional SDS-PAGE pattern (1DE). More spots rather than protein bands (19 versus 11) were visualized by 2DE and 1DE, respectively, suggesting the presence of either protein isoforms or posttranslational modifications. Sera of mice and hamsters immunized through exposure to sand fly bites following different immunization schedules showed elevated anti-saliva IgG levels. These sera allowed the detection of 5 bands and 16 immunogenic spots in 1DE and 2DE, respectively, followed by WB. These antigens were identified by MALDITOF/TOF as SP03, SP03B, SP08, SP01, SP01B, SP04, SP04B, SP02, Phlebotomus ariasi SP16, and Phlebotomus argentipes SP13. This work is assumed to be the first attempt to establish 2DE proteomic maps of P. perniciosus saliva. All spots were identified as salivary proteins, confirming this technology as an interesting tool to improve sand fly salivary knowledge.