Zoonotic pathogens that can be transmitted via food to humans have a high potential for large-scale emergencies, comprising severe effects on public health, critical infrastructures, and the economy. In this context, the development of laboratory methods to rapidly detect zoonotic bacteria in the food supply chain, including high-resolution mass spectrometry are needed. In this work, an optimized sample preparation method for liquid chromatography-mass spectrometry (LC-MS) based proteome profiling was established for Francisella isolates and a cluster analysis, as well as a phylogenetic tree, was calculated to shed light on evolutionary relationships. Furthermore, this method was applied to tissues of infected hare carcasses from Germany. Even though the non-informative data outnumbered by a manifold the information of the zoonotic pathogen in the resulting proteome profiles, the standardized evaluation of MS data within an established automated analysis pipeline identified F. tularensis and thus is in principle an applicable method to monitor food supply chains.