In veterinary diagnostic laboratories identification of mycoplasmas is achieved by demanding, cost-intensive and time-consuming methods that rely on antigenic or genetic identification. Since MALDI-ToF MS seems to represent a promising alternative to the currently practiced cumbersome diagnostics we assessed its applicability for the identification of almost all mycoplasma species isolated from vertebrate animals so far. For generating main spectrum profiles (MSPs) the type strains of 98 Mycoplasma , 11 Acholeplasma , and 5 Ureaplasma species, and in case of 69 species, one up to 7 clinical isolates were used. To complete the database, 3-7 representatives of 23 undescribed Mycoplasma species isolated from livestock, companion animals and wildlife were also analyzed. A large in-house library containing 530 MSPs was generated and the diversity of spectra within a species was assessed by constructing dendrograms based on a similarity matrix. All strains of a given species formed cohesive clusters clearly distinct from all other species. In addition, phylogenetically closely related species also clustered closely but were separated accurately indicating that the established database was highly robust, reproducible and reliable. Further validation of the in-house mycoplasma library using 335 independent clinical isolates of 32 mycoplasma species confirmed the robustness of the established database by achieving reliable species identification with log scores ≥ 1.80. In summary, MALDI-ToF MS proved to be an excellent method for the identification and differentiation of animal mycoplasmas, combining convenience, ease, speed, precision and low running costs. Furthermore, this method is a powerful and supportive tool for the taxonomic resolution of animal mycoplasmas.