Background <p>Fast atom bombardment-mass spectrometry (FAB-MS) measurement using a thiol-containing matrix such as α-thioglycerol (TG) can, in principle, determine the number (<i>n</i>) of disulfide bonds in cyclic oligopeptides (COPs) by detecting reduced ions of the type [M+<i>n</i>2H + H]⁺.</p> Findings <p>Although the FAB-MS using TG (TG method) successfully generated the reduced ions [M+ <i>n</i>2H + H]⁺ for simple COPs such as oxytocin and its homologous peptides, it failed to produce the corresponding ions for urotensin II (<i>n</i> = 1), guanylin (<i>n</i> = 2), and linaclotide (<i>n</i> = 3). In this study, we demonstrated that performing TG reduction on a thin-layer chromatography (TLC) plate, where silica-gel accelerates the reduction, followed by FAB-MS analysis (TG/TLC method), effectively yields [M+<i>n</i>2H + H]⁺ or [M+ <i>n</i>2H + Na]⁺ for disulfide-linked COPs with <i>n</i> = 1 − 3.</p> Conclusion <p>As an alternative approach, FAB-MS using dithiodiethanol (DTDE) as the matrix readily provides the intact [M + H]⁺ ion of disulfide-containing COPs. Therefore, comparing FAB-MS using DTDE with the TG/TLC method offers a reliable strategy for determining the number of disulfide bonds in COPs.</p> Graphical Abstract <p></p>