Recent EU directives and regulations for quality control and authentication of food products have prompted the development of new methods for large-scale tests to ensure the protection of consumers. In view of this, an innovative method based on MALDI-TOF mass spectrometry has been developed and successfully applied to fish authentication. Highly specific mass spectrometric profiles from 25 different fish species were obtained. Signals generated from proteins with molecular weights of about 11 kDa have been selected as specific biomarkers for unambiguous discrimination. This method is also suitable for verifying commercial product authenticity and to rapidly discriminate species subjected to fraudulent substitutions, such as those belonging to Gadidae and Pleuronectiformes. For example, biomarkers for fillets of sole (m/z 11975.21), European plaice (m/z 11351.73, 11763.63) and Greenland halibut (m/z 11432.38) were defined. Structural characterization by mass spectrometry of several proteins generating biomarker signals allowed us to identify them as parvalbumins, known to be among the major fish allergens.