4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and cotinine are two efficient biomarkers of tobacco-specific carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and nicotine. In this contribution, we developed an effective system of two-dimensional liquid chromatography coupled with tandem mass spectrometry for high-throughput simultaneous determination of NNAL and cotinine in smoker’s urine. The strong cation-exchange (SCX) and the reversed-phase chromatography (RPLC) were adopted in the first and second dimensional studies. As mentioned below, the two-dimensional system is discussed. The orthogonality of this two-dimensional system was evaluated through the separation of NNAL and cotinine. In 2D-SCX/nRPLC, NNAL and cotinine can be fractionated in the same dimension (SCX: 5 µm, 35 mm × 2.0 µm i.d.) by step elution, followed by the molecular separation in the second dimension (RP: 5 µm, 150 mm × 4.6 µm i.d.) with binary gradient LC. In this work, the limits of detection of the method were 0.24 pg/mL and 0.054 ng/mL for NNAL and cotinine respectively. The recoveries of the spiked samples were 99.8 %-105.1 % and 111.9 %-114.2 % for NNAL and cotinine in urine, respectively. The precisions of the method were 0.43 %-3.07 % and 0.73 %-2.11 %. The method is useful for monitoring NNAL and cotinine in smoking and satisfactory to evaluate tobacco exposure.