Mass spectrometry for rapid identification of microorganisms is expanding over the last years because this approach is quick. This methodology provides a decisive interest to fight against bioterrorism as it is applicable whatever the pathogen to be considered and often allows subtyping which may be crucial for confirming a massive and widespread attack with biological agents. Here, we present a methodology based on next-generation proteomics and tandem mass spectrometry for discovering numerous protein biomarkers allowing the discrimination of spores and vegetative cells of Bacillus atrophaeus, a biowarfare simulant. We propose a global quantitative evaluation of the two groups of discriminant biomarkers based on their aggregated normalized spectral abundance factors.