Culicoides biting midges are of great importance as vectors of pathogens and elicitors of allergy. As an alternative for the identification of these tiny insects, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was evaluated. Protein mass fingerprints were determined for 4-5 field-caught reference (genetically confirmed) individuals of 12 Culicoides species from Switzerland, C. imicola from France, laboratory-reared C. nubeculosus and a non-biting midge. Reproducibility and accuracy of the database was tested in a validation study by analysing 108 mostly field-caught target Culicoides midges and 3 specimens from a non-target species. A reference database of biomarker mass sets containing between 24 and 38 masses for the different species could be established. Automated database-based identification was achieved for 101 of the 108 specimens. The remaining 7 midges required manual full comparison with the reference spectra yielding correct identification for 6 specimens and an ambiguous result for the seventh individual. Specimens of the non-target species did not yield identification. Protein profiling by MALDI-TOF, which is compatible with morphological and genetic identification of specimens, can be used as an alternative, quick and inexpensive tool to accurately identify Culicoides biting midges collected in the field.