The accurate identification of individuals in zooplankton samples is a crucial step in many plankton studies. Up to now, this has been done primarily by microscopic analysis of morphological characters, and new molecular methodologies are still relatively rarely applied. Another promising technology is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which has had a major impact in applied and systematic microbiology, where it is used for routine high throughput identification of bacteria and fungi. For the present study, we developed a protocol for the rapid acquisition of mass spectra from whole individual copepods. The final protocol enabled us to obtain mass spectra with more than 100 distinct peaks in the mass range of 2000–20 000 Da. A comparison of the mass spectra of three species of Eudiaptomus showed that they could all be clearly discriminated, whereas the mass spectra of different developmental stages and sexes of each particular species were highly similar. Further, a discrimination of con-specific individuals from different habitats was achieved, at least partly, even without extensive optimization of the analytical and statistical procedures. These results indicate the feasibility of identifying copepods by a rapid and simple MALDI-TOF MS analysis, e.g. for population ecology studies.