Brucella canis is pathogenic for dogs and humans. Serological diagnosis is a cost-effective approach for disease surveillance, but a major drawback of current serological tests is the cross-reactivity with other bacteria that results in false positive reactions, and development of indirect tests with improved sensitivity and specificity remain a priority. A western blotting assay was developed to define the serum antibody patterns associated to infection using a panel of positive and negative dog sera. B. canis positive sera recognized immunogenic bands ranging from 7 to 30 kDa that were then submitted to ESI–LC-MS/MS and analyzed by bioinformatics tools. A total of 398 B. canis proteins were identified.. Bioinformatics tools identified 16 non cytoplasmic immunogenic proteins predicted as non-homologous with the most important Brucella cross-reactive bacteria and 9 B. canis proteins non-homologous to B. ovis; among the latter, one resulted non-homologous to B. melitensis. The western blotting test developed was able to distinguish between infected and non-infected animals and may serve as confirmatory test for the serological diagnosis of B. canis. The mass spectrometry and in silico results lead to the identification of specific candidate antigens that pave the way for the development of more accurate indirect diagnostic tests.