A simple, sensitive, selective, and reproducible method based on high-performance liquid chromatography-electrospray ionizationtandem mass spectrometry was developed for the determination of nicotine and its major metabolite cotinine in human urine. The internal standard (acetaminophen) was separated from cotinine on a Hypersil Gold C18 column with retention times of 9.3 and 13.0 min, respectively. The mobile phase consisted of a mixture of 10 mM acetate buffer (pH 5.4) and methanol (45:55, v/v), running through the column at a flow rate of 0.6 mL/min. The chromatographic analysis was operated at 25°C. Sample preparation was prepared by liquid-liquid extraction with a mixture of methyl-t-butyl ether in dichloromethane (1:1, v/v). The precision of the method based on within-day repeatability and reproducibility (day-to-day variation) was below 15% (% coefficient of variation). Good accuracy was observed for both the intra-day or inter-day assays. Limit of quantification was accepted as 0.02 ng using 100 mL samples. The mean recoveries for cotinine and the internal standard were greater than 90%. The method has been applied to the investigation of a 2-h urinary excretion of cotinine in 154 healthy non-smoking Thai volunteers (aged 18-45 years) following the administration of a half-piece (2 mg) of nicotine gum.