KR-31831 ((2S,3R,4S)-4-(((1H-imidazol-2-yl)methyl)(4-chlorophenyl)amino)-6-amino-2-(dimethoxymethyl)-2-methyl-3,4-dihydro-2H-chromen-3-ol) is a novel antiangiogenic agent. In vitro and in vivo metabolism of KR-31831 in rats has been investigated using LC-MS and LC-MS/MS analysis. Incubation of rat liver microsomes and hepatocytes with KR-31831 produced three metabolites (M1-M3). M1, M2, and M3 were identified as N-((1H-imidazol-2-yl)methyl)-4-chlorobenzenamine, (2R,3R,4S)-4-(((1H-imidazol-2-yl)methyl)(4-chlorophenyl) amino)-6-amino-2-(hydroxymethyl)-2-methyl-3,4-dihydro-2H-chromen-3-ol, and N-((2S,3R,4S)-4- (((1H-imidazol-2-yl)methyl)(4-chlorophenyl)amino)-2-(dimethoxymethyl)-3-hydroxy-2-methyl-3,4-dihydro-2H-chromen-6yl)acetamide, respectively, by co-chromatography with the authentic standards and by comparison with product ion spectra of the authentic standards. Those in vitro metabolites were also detected in bile, plasma, or urine samples after an intravenous administration of KR-31831 to rats. The metabolic routes for KR-31381 included the metabolism of acetal group to hydroxymethyl group (M2), N-dealkylation to M1, and N-acetylation at the 6-amino group (M3).