Abstract. Helium direct analysis in real time (He-DART) mass spectrometry (MS) of some compounds, polysaccharides, for example, usually tends to be challenging because of the occurrence of prominent in-source decay (ISD), which was considered as an undesired side reaction, as it complicated the resulting mass spectra. Our approach is to take advantage of an efficient and practical method termed the temperature-dependent ISD (TDISD) technique combined with fragmentation of the dehydrated dimers using DART Q-TOF tandem mass spectrometry for differentiation of disaccharide isomers. In this study, cross-ring cleavages and non-ovalent complexes were detected in the spectra of the saccharides. It was observed that the gas heater temperature had a significant effect on the absence or presence of signal in DART spectra. At high gas temperature, ions in high mass region began to appear. Based on the types of crossring cleavages and noncovalent complexes, disaccharide isomers with different linkage positions can be differentiated in both positive and negative ion modes at a lower DART gas temperature. Additionally, anomeric configurations were assigned on the basis of the relative abundance ratio of m/z 198:342 obtained by the comparison of the positive ion mode tandem mass spectrum of an α isomer dimer generated at higher DART gas temperature and that of the corresponding β one. In general, this method is easy, fast, effective, and robust for identifying disaccharide isomers.