Abstract: Differentiation of isobaric glutamine (Gln) and lysine (Lys) is needed for complete de novo sequencing and require high resolution mass spectrometry generally. Both Gln and Lys can produce immonium ions at m/z 101 and their related ions at m/z 84. Although it is known that the peak at m/z 101 due to Lys is sometimes weak and abundant ions at m/z 84 is a good indicator of the presence of Lys, whether this feature can be used to discriminate Lys and Gln is still a controversial issue. In this study, investigations were used to distinguish Gln and Lys based on immonium and immonium-related ions produced by matrix-assisted laser desorption ionization tandem time-of-flight (MALDI-TOF/TOF) with high energy collision induced dissociation (CID). The work focused on a series of synthetic peptides and 184 tryptic peptides. The result shows that Gln is more prone to generate immonium ions at m/z 101comparede to Lys, which generates m/z 84 and m/z 129 ions mainly. It is the ratios of intensity of m/z 101 ion versus m/z 84, rather than the intensity value of them, can be used to distinguish Gln versus Lys for practical samples for those peptides that containing Lys or Gln, or provide clue for the number and position of Gln for Lysterminated peptides. Additionally, the mass measurement at m/z 101 is also relevant for distinguishing Lys and Gln. This observation has demonstrated that the usefulness of low mass region information of TOF/TOF spectra in distinguishing isobaric Lys and Gln.