Objective To evaluate matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for detection of pathogenic bacteria in laboratory animals. Methods In total, 40 standard strains and 384 laboratory animal isolates were identified using the Bruker Biotyper system. Results The accuracy rates of Bruker Biotyper to detect standard strains and isolates were 90. 0% and 80. 0%, respectively. There were biases in the identification of Salmonella typhimurium, Yersinia pseudotuberculosis, Shigella spp., Klebsiella pneumoniae, Pasteurella pneumotropica biotype Heyl, and Bordetella bronchiseptica. Conclusions The MALDI-TOF MS method is highly efficient and accurate. It can be used for rapid identification of pathogenic bacteria in laboratory animals