Dihydromyricetin (DMY) is a health-beneficial flavanonol in the family of flavonoids, extracted from Ampelopsis grossedentata (Hand.-Mazz.) W. Wang. When investigating the stability of DMY in Dulbecco's modified Eagle's medium (DMEM) solution at 37°C by LC-MS/MS, four nitrogenous derivatives of DMY were found except for the previously known dimers, oxidized products, and ring-cleavage products of DMY. Compound 1 (N-DMY) was identified to be 4′-amino (NH₂)-DMY based on MS and NMR data. The formation mechanism of N-DMY was thought to include two steps. First, the pyrogallol moiety on the ring B of DMY was prone to be oxidized to quinone. Second, quinone might further participate in the Strecker degradation of amino acids in DMEM to form N-DMY and corresponding aldehydes. Correspondingly, compounds 2–4 might be formed by the Strecker degradation reaction of amino acids in DMEM with quinones from the oxidation of DMY dimers and DMY ring-cleavage products. Consistent with the formation mechanism, it was observed that flavonoids with a catechol or pyrogallol moiety readily form their nitrogenous derivatives during the incubation in DMEM at 37°C, which might be because catechol or pyrogallol are easily oxidized to quinones. In addition, the nitrogenous derivatives of flavonoids may have a potential to participate in the metabolism of flavonoids during in vitro and in vivo experiments. Therefore, flavonoid nitrogenous derivatives may play a significant role in the actual effects of flavonoids, which need to be further investigated in the future.