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Background: The diagnosis of brucellosis by serological tests is based on antigen suspensions derived from smooth lipopolysaccharide extracts, which can give false positive results linked to cross-reactivity with other Gram-negative microorganisms, especially Yersinia enterocolitica O:9 and Escherichia coli O157:H7. Objective: The objective of the present study was the characterization by proteomic analysis of specific immunogenic proteins not associated with smooth lipopolysaccharide to improve the diagnostic tests used in the ovine brucellosis eradication programs. Methods: The serum from a sheep positive to Brucella melitensis was treated to eliminate all antibodies against such lipopolysaccharide and highlight the reaction towards the immunoreactive proteins in Western Blotting. Results: The immunoreactive bands were identified by nLC-MS/MS and through bioinformatic tools, it was possible to select 12 potential candidates as protein antigens specific for Brucella melitensis. Conclusion: The detection of new antigens not subjected to cross-reactivity with other Gram-negative microorganisms can offer an additional tool for the serological diagnosis of such disease.