Currently used techniques for species identification are mostly relied on protein database matching or alternatively on immunological procedures. Although widely used, such an approach does not always provide satisfactory matching, sequence coverage or specific antigen-antibody reaction to unambiguously identify DNA, RNA, lipid, and sugar or peptide/protein of selected species. Mass spectrometry as a tool for species determination was introduced about ten years ago. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identifies microbes using either intact cells or cell extracts. The most abounded proteins from the cell detected in the instrument are matched against commercially available databases that cover relatively limited number of microbes. On the other hand, high resolution mass spectrometers in contrast to MALDI-TOF MS could provide more accurate and precise results after de novo sequencing of analyzed proteins.