RATIONALE: N-[2-Hydroxy-3,5-dimethylbenzilidene]-Hup A (H14) is a derivative of huperzine A (Hup A) that demonstrates superior protective effects against soman (GD) compared to Hup A. This study aims to evaluate the protective efficacy of H14 pretreatment against GD in rats and to provide an analytical framework for the pharmacokinetic evaluation of H14 in experimental animals. METHODS: The study employed protective ratios (PR) as an evaluation criterion to assess the efficacy of H14 and Hup A in preventing GD in vivo. Liquid-liquid extraction techniques were utilized to extract H14 and its metabolite, Hup A, from plasma. The extracted plasma samples were then analyzed using an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous quantification of H14 and Hup A. RESULTS: The PR values for the 6- and 12-h Hup A groups were 1.26 and 1.08, respectively. In contrast, the 6, 12, and 24-h H14 groups demonstrated PR values of 2.81, 1.98, and 1.18, respectively, indicating extended protective capabilities compared to Hup A. All validation parameters for the UHPLC-MS/MS method, including linearity, specificity, precision, accuracy, matrix effect, and stability, met the acceptance criteria established by FDA guidelines. The pharmacokinetic analysis indicates that H14, after conversion to Hup A in vivo, significantly extends the duration of Hup A concentrations in the body, leading to more effective prevention of GD poisoning compared to Hup A alone. CONCLUSIONS: H14 demonstrates superior efficacy in preventing GD poisoning compared to Hup A. Furthermore, this analytical approach offers a reliable and efficient method for the pharmacokinetic evaluation of H14 in experimental animals.