In terms of the development of new medicines, the identification of peptides in organs and tissues is crucial. Because time-of-flight secondary ion mass spectrometry (TOF-SIMS) has extremely high sensitivity and does not require a special pretreatment of samples, it is one of the most promising analysis techniques for evaluation of unknown peptides in organ extract. It is, however, often difficult to interpret TOF-SIMS spectra of biomolecules because of the overlap of fragment ions from biomolecules and organic molecules and the difficulty for identifying high mass secondary ions. Therefore, appropriate data analysis methods such as G-SIMS and multivariate analysis are necessary to characterize intricate TOF-SIMS spectra. In this study, G-SIMS was applied to interpret TOF-SIMS spectra of peptide samples. As a result, molecular ions of two peptides, approximately 400 molecular weight, were indicated automatically using G-SIMS analysis without preinformation on the tested peptides. Copyright © 2012 John Wiley & Sons, Ltd.