4,5-Dimethoxycanthin-6-one and 5-hydroxy-4-methoxycanthin-6-one are the active ingredients of P. quassiodes. In the present work, a LC-MS/MS method was developed for the determination of 4,5-dimethoxycanthin-6-one and its major metabolites 5-hydroxy-4-methoxycanthin-6-one (M1) and 4-hydroxy-5-methoxycanthin-6-one (M2) in rat plasma and tissues, and applied to study their pharmacokinetics and tissue distribution after intramuscular administration of 4,5-dimethoxycanthin-6-one to rats. By protein precipitation with methanol for plasma samples and liquid-liquid extraction with ethyl acetate for tissue samples, the analytes were separated on an ODS C18 column with a mobile phase consisted of methanol and water (0.1% formic acid), and quantified by a MS detector in positive multiple reaction monitoring (MRM) mode. MS transitions were m/z 281.0 → 167.1 for 4,5-dimethoxycanthin-6-one, m/z 267.0 → 168.1 for M1 and M2, m/z 251.0 → 195.1 for 3-methylcanthin-2,6-dione (IS). The pharmacokinetic results indicate that 4,5-dimethoxycanthin-6-one is absorbed rapidly (Tmax = 5.4–6.4 min), distributed rapidly and widely in the order of liver > kidney ≈ lung ≈ large intestine ≈ small intestine, and eliminated quickly (t1/2z = 64.9–77.7 min) following the intramuscular administration. Furthermore, M1 and M2 were detected only in rat plasma and liver at the indicated times after the intramuscular administration.