A method based on liquid chromatography tandem mass spectrometry (LC-MSMS) was developed for the direct determination of nicotine, cotinine, trans-3'-hydroxycotinine, their corresponding glucuronide conjugates, as well as nornicotine, norcotinine, cotinine-N-oxide and nicotine-N'-oxide in the urine of smokers. The assay involves centrifugation and filtration of diluted urine, chromatography was performed on a C18 reversed-phase column using a gradient of 10 mM ammonium acetate, pH 6.8, and methanol as a mobile phase at a flow rate of 1 mL/min. Separated analytes were determined by electrospray ionization tandem mass spectrometry in the positive ion mode of multiple reaction monitoring. Deuterium-labeled nicotine, cotinine, and trans-3'-hydroxycotinine were used as internal standards. Calibration curves were linear over the calibration ranges for all the substances under investigation, with r2 > 0.998. Precision (RSD) for all the analytes at high, medium and low levels was between 2.1% and 17%. Recoveries of nicotine and nine of its major metabolites ranged from 78 to 116%. The described LC-MSMS method allows the simultaneous determination of nicotine and nine of its major metabolites in the urine of smokers with good precision and accuracy. Since the method requires a simpler sample pre-treatment and a short time for chromatography (8 min), it is suitable for determining the nicotine dose in mass human biomonitoring studies.