Abstract: A high performance liquid chromatographyelectrospray ionization tandem mass spectrometry （LCESIMS/MS）method was developed for the determination of cotinine in urine. After dilution, centrifugation and filtration, the urine was injected to the LC-MS/MS. The analysis was performed on a Agilent Zorbax Eclipse XDB-C₁₈ column（150 mm×2.1 mm, 3.5 μm） using a gradient elution with the mobile phases of 0.1% formic acid buffer and 0.1% acetate in methanol (V/V), and determined by tandem mass spectrometry in positive ESI mode under multiple reaction monitoring mode. The limits of determination (S/N=3) was 0.08 μg/L, with a linear calibration range spanning 1—500 μg/L. The recoveris ranged from 94.3% to 99.9% at three spiked levels, the relative standard deviations (RSDs) ranged from 0 to 3.2%. The method was applied to detect the concertration of urine cotinine from 58 non-smokers and 246 smokers. Twenty-four hours urine cotinine levels in smokers was much more than 180 times that in non-smokers. However, due to the differences between individuals and smoking behavior, the concertration of urine cotinine did not increase with cigarettes nicotine in the mainstream smoke.