Matrix assisted laser desorption/ionisation - time of flight mass spectrometry (MALDI-TOF MS) is a rapid, accurate and sensitive technique for providing molecular weight information of compounds like proteins, peptides, oligonucleotides, nucleic acids and carbohydrates. Its relative high tolerance towards contaminations (e.g. salts), makes it a valuable technique, complementary and competitive to other mass spectrometric techniques having other ionisation principles. The mass accuracy of the UltraFlextreme is 1 - 5 ppm and the mass resolution goes up to 40000 in reflector mode. The range of molecular weights, which can be analysed by routine with MALDI-TOF MS varies from ca. 100-150.000 Da, although in rare cases proteins with a Mw above 1 MDa have been analysed successfully. With the UltraFlextreme it is also possible to perform MS/MS on every peak in the spectra using the TOF/TOF option. Virus detection and/or identification traditionally rely on methods based on cell culture, electron microscopy and antigen or nucleic acid detection. These techniques are good, but often expensive and/or time-consuming; furthermore, they not always lead to virus identification at the species and/or type level. In this study, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) was tested as an innovative tool to identify human polioviruses and to identify specific viral protein biomarkers in infected cells. The results revealed MALDI-TOF MS to be an effective and inexpensive tool for the identification of the three poliovirus serotypes. The method was firstly applied to Sabin reference strains, and then to isolates from different clinical samples, highlighting its value as a time-saving, sensitive and specific technique when compared to the gold standard neutralization assay and casting new light on its possible application to virus detection and/or identification.