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Traditional methods to identify bacterial pathogens involved in fish diseases are often slow and/or costly. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS) is an established, fast, accurate and cost effective protein-based technique for identification of bacterial pathogens in human and veterinary laboratories, and has now also been developed for identification of bacterial fish pathogens. Based on analysis of colonies sampled directly from agar plate cultures, the MALDI-TOF technique generates a protein spectrum which is then compared against spectra, called reference spectra, stored in the MALDI-TOF databases. Since not all bacterial fish pathogens are covered in commercial databases, database-libraries are usually constructed locally for relevant reference strains/species. In this study, results from two collaborative projects, involving four European veterinary medicine institutes, are presented, focusing on supplementation of available spectra and validation of the MALDI-TOF MS technique for identification of several bacterial fish pathogens. Flavobacterium psychrophilum, F. columnare, Pseudomonas anguilliseptica, Renibacterum salmoninarum, Vibrio anguillarum and Yersinia ruckeri are examples of important bacterial fish pathogens that may be successfully identified to the species level by MALDI-TOF MS. On the contrary, identification of Aeromonas salmonicida to species level requires additional phenotypic or genetic analysis to arrive at a correct identification. In conclusion, MALDI-TOF MS has proven a fast, cost effective and accurate identification method for use in fish bacteriology, with a potential for further improvements.