Determination of nicotine metabolites in human urine provides an ideal tool for the assessment of the tobacco use-related nicotine exposure， provided that the considered metabolites comprise a large share of the amount exposed. Nicotine and its nine metabolites mentioned in this research comprised of more than 90% of the total nicotine metabolites. A hydrophilic interaction high performance liquid chromatography－tandem mass spectrometry was developed for the simultaneous determination of nicotine and its nine metabolites in human urine. The urine sample was diluted 20 times with a mixed solvent of acetonitrile and methanol（3∶1， by volume）， and subsequently filtered through a 0.22 μm membrane for direct analysis. Then these metabolites were separated on an Atlantis HILIC Silica column（3.0 mm × 100 mm， 3.0 μm） by gradient elution， with 10 mmol/L ammonium formate aqueous solution（adjusted to pH 3.0 with formic acid）－acetonitrile as mobile phases， then detected by tandem mass spectrometry with electrospray ionization in positive ion mode under multiple reaction monitoring（MRM）， and finally quantified by isotope-labelled internal standards. Results showed that the calibration curves for nicotine and its nine metabolites were linear in the certain concentration range with correlation coefficients（,r,） larger than 0.997. The limits of detection（LOD） and quantitation（LOQ） were in the ranges of 0.03－0.24 ng/mL and 0.10－0.80 ng/mL， respectively. The recoveries at low， medium and high spiked levels in real samples ranged from 81.9% to 110%， with intra-day and inter-day relative standard deviations of 0.50%－6.7%. The developed method could meet the requirements for high-throughput detection of nicotine and its nine metabolites in human urine with simplicity， high sensitivity and stability.