To establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of levomethorphan, dextromethorphan and its metabolite dextrorphan in hair and urine, hair samples were ground and extracted by ultrasonication using a methanol solution containing SKF525A as the internal standard, while urine samples were processed by protein precipitation with the internal standard diazepam-D5. An Astec® CHIROBIOTIC® V2 column (250 mm × 2.1 mm, 5 µm) was used with the mobile phase consisting of 10 mmol/L ammonium acetate containing 0.5‰ formic acid and methanol. Detection was performed using positive electrospray ionization in multiple reaction monitoring mode. The linearity for levomethorphan, dextromethorphan, and dextrorphan in hair samples was 0.1–10 ng/mg, the limits of detection (LOD) were 0.05 ng/mg, and the limits of quantification (LOQ) were 0.1 ng/mg. In urine samples, the linearity was 1–200 ng/mL, LOD was 0.5 ng/mL, and LOQ was 1 ng/mL. Method validation met the requirements for biological sample testing and can be applied to drug monitoring. In actual case samples from China, dextromethorphan and dextrorphan were detected, while levomethorphan was not observed.This method is simple, fast, and highly sensitive to be used for the qualitative and quantitative analysis of levomethorphan, dextromethorphan, and dextrorphan in hair and urine samples.