Dravet syndrome, also known as severe myoclonic epilepsy of infancy (SMEI), is a rare childhood epileptic encephalopathy with limited effective treatment options. Previously, we identified a novel 1,3,4-oxadiazol-2(3H)-one derivative, GM-91466, which demonstrated potent antiseizure efficacy by upregulating brain serotonin (5-HT) levels in a mouse model. However, a validated bioanalytical method and pharmacokinetic characterization for GM-91466 have not yet been established. In this study, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was established for the quantitative analysis of the GM-91466 and applied it to pharmacokinetic evaluation in biological samples. Plasma samples were prepared by protein precipitation using acetonitrile with a structurally similar compound (GM-91007) as the internal standard. The method showed good linearity over the range of 1–1000 ng/mL (r² > 0.99) and met acceptance criteria for accuracy and precision across all quality control levels. The validated method was applied to pharmacokinetic studies in mice, rats, and monkeys, enabling reliable determination of key parameters such as C_max, T_max, and AUC. In addition, in vitro pharmacokinetic evaluations including phase I metabolic stability, plasma protein binding, and plasma stability were performed. Overall, this LC-MS/MS-based bioanalytical platform enables reliable pharmacokinetic evaluation of GM-9146 and supports further preclinical development for Dravet syndrome