Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has shown promise in species identification of insect species. We evaluated its potential to consistently characterize laboratory-reared biting midges of the species Culicoides nubeculosus (Meigen) (Diptera: Ceratopogonidae). Twenty-one reproducible potential biomarker masses for C. nubeculosus were identified under different experimental treatments. These treatments included the homogenization of insects in either water or known concentrations of formic acid. The biomarker masses were present independent of age, gender and different periods of storage of individuals in 70% ethanol (a standard preservation method). It was found that the presence of blood in females reduced the intensity of the MALDI-TOF pattern, necessitating the removal of the abdomen before analysis. The protein profiles of a related non-biting midge, Forcipomyia sp. (Diptera: Ceratopogonidae), and of Aedes japonicus japonicus (Theobald) (Diptera: Culicidae) mosquitoes were also examined and were distinctly different. These findings provide preliminary data to optimize future studies in differentiation of species within the Culicoides genus using MALDI-TOF MS which is a rapid, simple, reliable and cost-effective technique.