Tobacco-specific nitrosamines (TSNAs) in cigarette smoke are known as potential carcinogens. 4-(methylnitrosamino)-l-(3-pyridine)-l-butanol (NNAL) and its glycoside derivates are established as biomarkers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) exposure in smokers and non-tobacco users. Many studies have been focused on the determination of TSNA biomarkers of exposure in urine, whilst venous blood, due to its complex matrix effects, was not frequently considered for analysis. The purpose of this study was to develop an efficient and robust (precise as well as accurate) method for the simultaneous analysis of NNAL, NNK, N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT), and N-nitrosoanabasine (NAB) in human plasma by using online solid phase extraction-liquid chromatography-tandem mass spectrometry (online SPE-LC/MS/MS). Target compounds were concentrated and purified from blood plasma using a two-dimensional online SPE procedure. The required performance in chromatographic separation of the target analytes was achieved by using an Atlantis T3 column. A two-dimensional SPE clean-up process for the plasma samples was used by applying a combination of a PRS cation exchange and resin GP reverse phase column. The combination of these SPE column types resulted in a significant reduction of matrix background in the sample, therefore the clean-up significantly improved the sensitivity and accuracy of the method. The limits of detection (LODs) of this method for the five TSNAs ranged from 0.04 to 0.09 pg/mL, which are much lower than those reported in literature. The developed method was validated by determining the free and total TSNA levels in the plasma of 52 non-smokers and 61 smokers. Our results showed that this method combines high sensitivity with high repeatability. Furthermore, the automated online SPE procedure offers high throughput at high accuracy as well as precision level.