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Mass spectrometry is a powerful analytical tool in biotechnology. The ‘soft’ ionization and desorption technologies matrix-assisted laser desorption/ionization and electrospray ionization have enabled mass spectrometric analysis of large biomolecules, such as proteins and nucleic acid amplification products, and paved the way for mass spectrometry to become a leading technology in current genomics and proteomics efforts. Large-scale analysis of single nucleotide polymorphisms by mass spectrometry has been commercially established. This article reviews applications of mass spectrometry for microsatellite analysis. Features and capabilities of the two most prominent techniques, matrix assisted laser desorption/ionization and electrospray-ionization mass spectrometry, are compared and their potential to address the limitations of conventional microsatellite analysis based on comparison of gel electrophoretic mobilities is explored.