Strychnine is an alkaloid with strong toxic properties. Poisoning results in muscular contractions and death through asphyxiation. Intentional or accidental poisonings with strychnine occur mainly in small animals, especially dogs and occasionally cats. Strychnine can be detected in the liver or stomach contents. Unfortunately, the determination of strychnine in these matrices, especially in postmortem examination, is subject to a significant matrix effect that makes it difficult to confirm the presence of the substance being determined. Therefore, we developed a new liquid chromatography method combined with mass spectrometry. One-gram homogenized samples were extracted and partitioned after adding acetonitrile and 5-mol solution of ammonium acetate. After extraction, the samples were analyzed using high-pressure liquid chromatography-MS/MS/MS. The results of validation fulfil the requirement of the confirmatory criteria according to SANTE/11945/2015 regarding apparent recoveries (98.97% to 104.0%), repeatability (2.9%–4.1%), and within-laboratory reproducibility (3.3%–4.6%). The method can be successfully applied to confirm strychnine poisoning cases.