Objective: To establish a method for the determination of strychnine in human serum by liquid chromatography tandem mass spectrometry. Methods: In April 2024, the serum samples were extracted using alkaline ethyl acetate. Separation was achieved using a Waters HSS T3 column (100 mm×2.1 mm, 1.8 μm) with a linear gradient elution program of water/acetonitrile (containing 5 mmol/L ammonium formate and 0.1% formic acid) as the mobile phase. The analyte was detected by using electrospray ionization (ESI) in the positive multiple reaction monitoring (MRM) mode and quantified using an internal standard method. Results: A good linearity was obtained in the concentration range of 0.01~20.0 μg/L for target compound with the correlation coefficients≥0.999. The limitof detection (LOD) and the limit of quantitation (LOQ) were 0.003 μg/L and 0.01 μg/L, respectively. The average spiked recovery was 101.3%, the intra-day precision was 2.8%-5.4%, and the inter-day precision was 3.9%-6.3%, respectively. Conclusion: Liquid chromatogrphy tandem mass spectrometry method exhibits high sensitivity, strong specificity, and good accuracy, and can be used for the determination of strychnine in Homo sapiens serum.