Protein primary structure elucidation is the key requirement in proteomics analyses. Except the Edman degradation, unambiguous amino acid sequence determination can be regularly performed by usage of tandem mass spectrometry, based on peptide ion fragmentation. Protein characteristics such as size, amino acid composition or post-translational modifications affect fragmentation. For ionization and fragmentation efficacy improvements, except chemical modification of peptides, fragmentation initiators can be used. Fragmentation initiators facilitate desolvation and generate gas phase radicals in the mass spectrometer. In this thesis, controlled in source peptide ion fragmentation induced by addition of fragmentation initiators was studied by means of matrix-assisted desorption/ionization mass spectrometry (MALDI-TOF/TOF MS). Fragmentation initiators used were classified as persulfates, peroxides, sulphones and nitriles. Radical mechanisms of peptide ion fragmentation mediated by fragmentation initiators were elucidated and confirmed by orthogonal experiment - chemically modified peptides fragmentation. Mass spectra obtained in the first stage mass spectrometry after fragmentation initiators addition are almost identical to conventional tandem mass spectra produced by collision induced dissociation. Product ions formed in ion source can be further fragmented in the second step of MS analysis. This concept enables multiple tandem mass spectrometry (pseudo MS3).