Multiple reaction monitoring (MRM) mass spectrometry (MS) assays were used to verify candidate protein biomarkers from two related studies of Brucella. In the first study, aimed at differentiating responders from non-responders to treatment for brucellosis, 228 peptides representing 48 markers identified during Discovery were tested on a new set of 190 samples collected before, during and after treatment. Thirty one of these biomarkers were confirmed to be differentially expressed in at least one of the comparisons considered. The goal of the second study was to verify if proteins originally identified in the secretome of a Brucella-infected macrophage model of infection and a trophoblast model of infection could be detected in serum of Brucella-infected patients and used as diagnostic biomarkers. The subset of qualified targets included in the MRM assay originated from the up-regulated proteins in each secretome. Seventy proteins represented by 149 peptides were selected from the macrophage infection and 123 proteins represented by 254 peptides for the trophoblast infection. The MRM assay could be developed in serum for 171 proteins, and was applied to 278 samples including patients infected with Brucella or other confounding diseases and their respective regional controls.