This study demonstrates that trypsin cleaves exclusively C-terminal to arginine and lysine residues. The research used a high-accuracy ion trap Fourier transform mass spectrometer to achieve over 100-fold increased confidence in peptide identification compared to typical ion trap experiments. The results show that non-tryptic peptides occur only as C-terminal peptides of proteins or as breakup products of fully tryptic peptides N-terminal to an internal proline. Simulating lower mass accuracy led to a large number of proteins being erroneously identified with non-tryptic peptide hits. The study concludes that such peptide hits in previous studies should be re-examined and that peptide identification should be based on strict trypsin specificity. Trypsin is a highly specific protease that cleaves C-terminal to arginine or lysine residues, leading to peptides in the preferred mass range for effective fragmentation by tandem mass spectrometry. This generally leads to informative high mass y-ion series and makes tandem mass spectra more easily interpretable. When analyzing peptide mixtures by liquid chromatography coupled to tandem mass spectrometry, a large number of fragmentation events occur. The tandem mass spectra are searched against amino acid sequence databases by database search algorithms. The identified peptides receive a score and are combined into lists of identified proteins. The study found that non-tryptic peptides are rare and mostly occur as C-terminal peptides of proteins or as breakup products of fully tryptic peptides. The results indicate that trypsin cleaves exclusively C-terminal to arginine and lysine. The study used a high-accuracy mass spectrometer to confirm this finding. The results have important implications for proteomics researchers seeking the optimal search parameters to minimize false positives. The study also highlights the importance of high mass accuracy in database searches for accurate peptide identification.This study demonstrates that trypsin cleaves exclusively C-terminal to arginine and lysine residues. The research used a high-accuracy ion trap Fourier transform mass spectrometer to achieve over 100-fold increased confidence in peptide identification compared to typical ion trap experiments. The results show that non-tryptic peptides occur only as C-terminal peptides of proteins or as breakup products of fully tryptic peptides N-terminal to an internal proline. Simulating lower mass accuracy led to a large number of proteins being erroneously identified with non-tryptic peptide hits. The study concludes that such peptide hits in previous studies should be re-examined and that peptide identification should be based on strict trypsin specificity. Trypsin is a highly specific protease that cleaves C-terminal to arginine or lysine residues, leading to peptides in the preferred mass range for effective fragmentation by tandem mass spectrometry. This generally leads to informative high mass y-ion series and makes tandem mass spectra more easily interpretable. When analyzing peptide mixtures by liquid chromatography coupled to tandem mass spectrometry, a large number of fragmentation events occur. The tandem mass spectra are searched against amino acid sequence databases by database search algorithms. The identified peptides receive a score and are combined into lists of identified proteins. The study found that non-tryptic peptides are rare and mostly occur as C-terminal peptides of proteins or as breakup products of fully tryptic peptides. The results indicate that trypsin cleaves exclusively C-terminal to arginine and lysine. The study used a high-accuracy mass spectrometer to confirm this finding. The results have important implications for proteomics researchers seeking the optimal search parameters to minimize false positives. The study also highlights the importance of high mass accuracy in database searches for accurate peptide identification.