Recently, mass spectrometry has become a powerful tool in cancer research. Mass spectrometry represents the method that allows identification, quantification and characterization of proteins in biological samples. Nowadays, it is mainly used for biomarker discovery that can enable early detection of cancer. This article is focused on protein analysis by mass spectrometry. At fi rst, mass spectrometry and its importance in proteomics are described. Subsequently ionization type and mass analyzers are discussed. This relates to the possibility of on-line or off -line analysis connection with separation techniques, such as liquid chromatography and electrophoresis. Diff erent approaches for preparing proteins and methods of analysis of biomolecules using mass spectrometers are described. In addition, the possibility of mass spectrometric analyses of samples and data processing are discussed.