Considering the huge potential of the technology MALDI-TOF MS in veterinary and food microbiology is still in its infancy. Characterization of bacteria with traditional methods in comparison to genetic analysis is still the predominant task performed in routine diagnostics. In this context, the reliable identification of pathogenic bacteria is of high relevance and the application of MALDI-TOF MS is a valuable addition to existing technologies. Furthermore, proteomic data have gained access to resolve taxonomy/phylogeny. In food microbiology the detection of zoonotic bacteria has a high priority and MALDI-TOF MS can already be applied on major food -borne bacteria, e.g. Campylobacter spp., Salmonella spp., Listeria spp. and E. coli. The diverse functions of lactobacilli and acetic acid bacteria, being essential for the fermentation process towards the spoilage of food products, prompted studies on these microorganisms. This resulted in the transformation of knowledge obtained from single bacteria to a microbial community in a certain product or production process. Independent of the organism and the targeted subject the establishment of biomarkers has a high priority for easy discrimination and identification. The increasing application of MALDI-TOF MS in routine veterinary or food microbiology asks for further development and extension of the technique which will be addressed in a separate chapter.