Diatom identification is based on classical morphological methods, focused on both the frustule shape and ornamentation analysis and possible chain-forming. Being many morphological aspects common to several species, diatom identification and characterisation is both time-consuming and high expertise demanding. Furthermore, the use of advanced microscopy, such as transmission electron microscopy (TEM) or Scanning Electron Microscopy (SEM) is frequently necessary to differentiate these organisms. In this work, the matrix-assisted laser desorption /ionisation time-of-flight mass spectrometry (MALDI-TOF MS) was used to differentiate diatom taxa in a preliminary study of the possibility of using this technique to obtain reliable fingerprinting of diatoms in the range 2 to 20 KDa. Seminavis robusta, Coscinodiscus sp., Thalassiosira sp. and Cyclotella meneghiniana were used. The influence of culture age was studied concerning reproducibility. It was shown that MALDI-TOF MS spectra varied with culturing time and the need of taking this in considerations when setting up experimental standard conditions in the identification of diatoms is clearly established. One of the most important observations was that each diatom strain presented a specific age from where the mass spectrum became reproducible. For all Seminavis robusta strains and for Cyclotella meneghiniana this age was 9 days whereas for Thalassiosira sp. and Coscinodiscus sp. this limit was 13 days. Even with the limitations related with culture aging versus spectra reproducibility, MALDI-TOF MS proved to be a useful complementary tool to identify and characterise diatoms at the level of genus. However, it seems that it was not possible to distinguish different mating types inside the same species, as evidenced by the results obtained with Seminavis robusta isolates.