High-resolution MS/MS spectra of peptides can be deisotoped to identify monoisotopic masses of peptide fragments. The use of such masses should improve protein identification rates. However, deisotoping is not universally used and its benefits have not been fully explored. Here, we developed MS2-Deisotoper, a tool for use prior to database search, to identify monoisotopic peaks in centroided MS/MS spectra. MS2-Deisotoper works by comparing the mass and relative intensity of each peptide fragment peak to every other peak of greater mass, and by applying a set of rules concerning mass and intensity differences. After comprehensive parameter optimisation, we show that MS2-Deisotoper could improve the number of peptide spectrum matches (PSMs) identified by up to 8.2% and proteins by up to 2.8%. It was effective with SILAC and non-SILAC MS/MS data. The identification of unique peptide sequences was also improved, increasing the number of human proteoforms by 3.7%. Detailed investigation of results showed that deisotoping increases Mascot ion scores, improves FDR estimation for PSMs and leads to greater protein sequence coverage. At a peptide level, we found that the efficacy of deisotoping was affected by peptide mass and charge. MS2-Deisotoper can be used via a user interface or as a command-line tool.