Background: Thailandepsin B (TDP-B) is a potent histone deacetylase inhibitor under development. A reliable bioanalytical method for the quantification of TDP-B in plasma samples is required. Results: The stabilizer mixture containing hydrochloric acid, formic acid and dichlorvos was applied to stabilize TDP-B in matrix samples. The method validation was conducted over the curve range of 1.00 to 1000 ng/ml. The intrabatch and interbatch precision and accuracy of the quality control samples showed ≤10.9% relative standard deviation and -6.7% to 15.0% relative error. Moreover, a possible metabolite M of TDP-B was tentatively characterized. Conclusion: A reliable LC-MS/MS method was developed and validated for the quantification of TDP-B and was successfully applied to a rat pharmacokinetic study.