Abstract: Differentiation of leucine (Leu) and isoleucine (Ile) is a difficult problem for de novo peptide sequencing. There is a general agreement that backbone cleavage is not capable of discriminating Leu and Ile residues and w-type ions produced by MALDI-TOF/TOF high energy collision-induced dissociation (CID) can provide distinguishable information. The experiment based on six pairs of tryptic-like isomeric peptides which contain 7 to 15 residues, showed that w ions were not always can be observed, while the displacement between Leu and Ile will lead to the change of relative abundance of immonium ions, internal fragment ions, b- and y-types of ions, the degree of changes depends on peptide composition and Leu/Ile position. Therefore, it is possible to distinguish Leu from Ile by comparing the relative intensities of the fragment ions of MALDI-TOF/TOF high energy CID spectra.