Flupirtine is well accepted as a centrally acting non-opioid analgesic with a favorable tolerability. During the stability study of flupirtine maleate drug product, an unknown degradation product (referred to as DP-I) exceeding the identification threshold was detected by gradient reverse phase HPLC method. To obtain this unknown impurity, the drug product was subjected to stress to enhance the level of DP-I. Furthermore, DP-I and three other thermal degradants (referred to as DP-II, DP-III and DP-IV resp.) were isolated by preparative HPLC. An isocratic preparative HPLC method was developed with a Welch Xtimate C18 column (250 mm × 30 mm, 5 μm) and the mobile phase composed of acetonitrile and 0.25 % ammonium hydroxide in water 70:30 (volume/volume). The flow rate was 20.0 mL min-1 and the chromatog. experiments were conducted at room temperature UV detection was carried out at 252 nm. Based on 1D-NMR, 2D-NMR and LC-MS spectral data, the structures of two novel degradation products were confirmed as di-Et 5-((4-fluorobenzyl)amino)-2-oxo-1H-imidazo[4,5-b]pyridine-1,3(2H)-dicarboxylate for DP-I and Et (2-(3,4-dimethyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6-((4-fluorobenzyl)amino)pyridin-3-yl)carbamate for DP-II. Moreover, the degradation mechanism from flupirtine maleate to DP-I and DP-II was also proposed.