Objective To create a high-performance liquid chromatography-tandem mass spectrometry (HPLCMS/MS) method for the quantitative analysis of Flavokawain B in plasma, and to investigate its pharmacokinetic characteristics in mice. Methods Flavokawain B was administered to KM mice via intravenous injection (20 mg/ kg), intraperitoneal injection (20, 40, and 60 mg/ kg), and intragastric(200, 400, and 600 mg/ kg) routes. Blood was drawn from the venous plexus of the eye fundus at 0, 5, 30 min and 1, 2, 4, 6, 8, 12, 16, and 24 h and then the plasma was separated. Flavokawain B levels in the plasma samples were analyzed by HPLC-MS/ MS, and its pharmacokinetic parameters and bioavailability following the different routes of administration were calculated. Results Flavokawain B showed a good linear relationship in plasma (r = 0. 9995), with a low limit of quantification of 0. 2 ng/ mL, accuracy -8. 50% ~ 12. 50%, precision 1. 73% ~ 12. 03%, and no obvious matrix effect (88. 68% ~ 102. 04%). HPLC-MS/ MS was verified as a suitable method for the quantitative evaluation of Flavokawain B in mice plasma. Flavokawain B showed rapid absorption in vivo, with a peak of plasma concentrations at 0. 083 h after intraperitoneal or intragastric administration. Flavokawain B showed good linear pharmacokinetics following intraperitoneal injection of 20 ~ 60 mg/ kg and intragastric administration of 200 ~ 600 mg/ kg. The absolute bioavailability of Flavokawain B was 53. 29% following intraperitoneal injection and 1. 38% following intragastric administration. Conclusions The current quantitative HPLC-MS/ MS technique provides a straightforward, precise, and sensitive method for investigating the pharmacokinetics of Flavokawain B in mice.