α-Amanitin and β-amanitin are highly toxic bicyclic octapeptides responsible for the poisoning of poisonous mush-rooms such as Amanita, Galerina, and Lepiota by inhibiting RNA polymerase II, DNA transcription, and protein synthesis. A sensitive, simple, and selective liquid chromatography-high resolution mass spectrometric method using parallel reaction moni-toring mode was developed and validated for the simultaneous determination of α- and β-amanitin in mouse plasma to evaluate the toxicokinetics of α- and β-amanitin in mice. Protein precipitation of 5 μL mouse plasma sample with methanol as sample clean-up procedure and use of negative electrospray ionization resulted in better sensitivity and less matrix effect. The calibra-tion curves for α- and β-amanitin in mouse plasma were linear over the range of 0.5–500 ng/mL. The intra- and inter-day coeffi- cient of variations and accuracies for α- and β-amanitin at four quality control concentrations were 3.1–14.6% and 92.5–115.0%, respectively. The present method was successfully applied to the toxicokinetic study of α- and β-amanitin after an oral adminis-tration of α- and β-amanitin at 1.5 mg/kg dose to male ICR mice.