In mollusc aquaculture, a large number of Vibrio species are considered as major pathogens that can cause high losses in hatchery and field. Thus, development of effective techniques for rapid detection and accurate identification of Vibrio involved in mortality events appears important to build efficient mollusc diseases surveillance programs. Phenotypic, biochemical and molecular techniques based on DNA amplification and sequencing are widely used for the identification of Vibrio species in the environment, but are time-consuming because of the use of different markers to differentiate closely related species. To provide the correct identification of unknown bacteria and species classification of environmental isolates, a tool is increasingly used, the matrix-assisted laser desorption/ionization time of flight spectrometry (MALDI-TOF), a proteomic method able to generate a specific proteomic bacteria profile in few seconds. Nevertheless, existing databases do not contain spectra for Vibrio associated with marine molluscs, consequently, we proposed to create a MALDI-TOF VibrioBase database containing containing 120 reference spectra of Vibrio species potentially responsible for molluscs diseases and belonging to 25 species: V. aestuarianus, V. cortegadensis, V. tapetis and species of Coralliilyticus, Harveyi, Mediterranei, Orientalis clades. A total of 72 mass proteomic spectra per strain cultured in three different media were generated and analyzed to determine specific reference spectra for each strain in order to perform the MALDI-TOF MS database specific to marine molluscs.To increase the specificity of the reference spectra, complementary statistical methods (PCA clustering and CCI matrix) were used. A perfect discrimination was obtained with all the created MSP of Vibrio species and even for very close related species. This VibrioBase is a first step that could enable the use of MALDI-TOF MS as a routine diagnostic tool for rapid bacterial identification in marine molluscs.This new database will be extended by including others strains of marine Vibrio as strains of Splendidus clade.