Dihydromyricetin has shown many bioactivities in cell level. However, dihydromyricetin was found to be highly instable in cell culture medium DMEM. Here, the underlying degradation mechanism was investigated via UPLC-MS/MS analysis. Dihydromyricetin was mainly converted into its dimers and oxidized products. At lower temperature, dihydromyricetin in DMEM showed higher stability. Vitamin C increased the stability of dihydromyricetin in DMEM probably due to its high antioxidant potential.