Brucellosis serodiagnosis is still a challenge and vaccination is the main measure used to control bovine brucellosis, being S19 and RB51 the most currently used vaccines. So, in order to contribute to brucellosis control, a bidimensional (2D) immunoblot-based approach was used to find immunogenic proteins to be used in serodiagnosis, particularly with ability to be employed in DIVA (Differentiating Infected from Vaccinated Animals) strategy. Immunoproteomic profile of Brucella abortus 2308 was analyzed in 2D western blotting using pooled sera from S19 vaccinated animals, RB51 vaccinated animals, B. abortus naturally infected animals and non-vaccinated seronegative animals. Evaluation of the antigens differentially immunoreactive against the groups of sera showed three proteins of particular importance: MDH (malate dehydrogenase) immunoreactive for S19-vaccinated animals, SOD (superoxide dismutase) reactive for infected animals and ABC transporter (multispecies sugar ABC transporter) reactive against sera from vaccinated animals (S19 and RB51). These three proteins were produced in E. coli and tested in an indirect ELISA (I-ELISA). For MDH, comparison between the vaccinated animals (independent of the vaccine used) and the seropositive and seronegative animals in I-ELISA showed significant differences. Data on the I-ELISA using SOD showed that sera from non-vaccinated naturally infected animals exhibited significant difference in comparison with all other groups. Otherwise, sera from vaccinated animals (S19 and RB51) and from non-vaccinated naturally infected animals did not show significant difference in OD values, but they were all significant different from non-vaccinated seronegative animals using ABC transporter as antigen in I-ELISA. In conclusion, together the 2D western blot analysis and the preliminary I-ELISA results suggest that the combined use of MDH and SOD could be successful employed in a LPS-free protein based serodiagnosis approach to detect bovine brucellosis and to discriminate vaccinated from naturally infected animals, in early post-vaccination stages.