This paper describes experience with the sequencing of synthetic segment peptides of gp41 which are used in trimeric peptides as potential peptide-based vaccines and as inhibitors to mimic the fusion state of gp41 in HIV infection using ESI/CID-FT-ICR MS. All b and y″ series ions are found in the CID spectra and the mass difference between the calculated and observed results is very slight. FT-ICR MS again shows its power in peptide sequencing, successfully helping us obtain the sequence of an unknown peptide.