We aimed to develop and validate a sensitive analytical method of nannozinone A, active metabolite of Nannochelins A extracted from the Myxobacterium Nannocytis pusilla, in mouse plasma using a liquid chromatography-tandem mass spec-trometry (LC-MS/MS). Mouse plasma samples containing nannozinone A and 13 C-caffeine (internal standard) were extracted using a liquid-liquid extraction (LLE) method with methyl tert-butyl ether. Standard calibration curves were linear in the concen-tration range of 1 - 1000 ng/mL (r 2 > 0.998) with the inter- and intra-day accuracy and precision results less than 15%. LLE method gave results in the high and reproducible extraction recovery in the range of 78.00–81.08% with limited matrix effect in the range of 70.56-96.49%. The pharmacokinetics of nannozinone A after intravenous injection (5 mg/kg) and oral administra- tion (30 mg/kg) of nannozinone A were investigated using the validated LC-MS/MS analysis of nannozinone A. The absolute oral bioavailability of nannozinone A was 8.82%. Plasma concentration of nannozinone A after the intravenous injection sharply decreased for 4 h but plasma concentration of orally administered nannozinone A showed fast distribution and slow elimination for 24 h. In conclusion, we successfully applied this newly developed sensitive LC-MS/MS analytical method of nannozinone A to the pharmacokinetic evaluation of this compound. This method can be useful for further studies on the pharmacokinetic opti-mization and evaluating the druggability of nannozinone A including its efficacy and toxicity.