Abstract: In order to accurately determine the tobacco-specific N-nitrosamines(TSNAs), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT) and N-nitrosoanabasine (NAB), in rabbit plasma, a solid-phase extraction column using tetraazacalix2arene2triazine modified silica (NCS) as sorbent was prepared. A solid-phase extraction/HPLC-MS/MS method was established by optimizing NCS solid-phase extraction conditions, chromatographic and mass spectrometric conditions. The results showed that NCS solid-phase extraction column was better than commercially available C₁₈ solid-phase extraction column in terms of the extraction efficiency of 5 TSNAs. The limits of detection were 0.0092-0.0590 ng/mL, and the mean recoveries for each analyte ranged from 90.1% to 113.3% with the relative standard deviation (RSD) of 0.95%-7.22%. This method is simple, accurate and suitable for the determination of TSNAs in rabbit plasma.