AbstractHeat stress studies have been conducted in support of developing a heat-stable liquid solution of dalbavancin. The degradation products that form in heat-stressed buffered dalbavancin solutions have been identified, including the known major degradation product, mannosyl aglycone (MAG), and four previously uncharacterized compounds. Liquid chromatography-mass spectrometry/mass spectrometry (LC–MS/MS) was used to identify the degradation products of dalbavancin in acetate- and phosphate-buffered solutions under thermal stress at 70 °C and the changes in the degradation pattern in the presence of 2HPβCD and divalent metal ions. Although Ca²⁺, Mg²⁺, and Zn²⁺ did not reduce dalbavancin degradation under thermal stress in acetate buffer, 2HPβCD significantly reduced its overall degradation, in particular, the formation of MAG. This protective effect was enhanced by the addition of Ca²⁺ to the formulation. In phosphate buffer, MAG formation was also reduced by the addition of 2HPβCD, although significant increases in other degradation products were observed in this case. The addition of Mg²⁺ to 2HPβCD significantly reduced the overall degradation while increasing MAG formation somewhat. The results strongly suggest that 2HPβCD forms a complex with the hydrophobic glycone tail of dalbavancin, suppressing hydrolysis of the glycosidic bond.