Abstract: In order to quantify four tobacco-specific N-nitrosamines (TSNAs) in sidestream cigarette smoke accurately, an online two-dimensional solid-phase extraction-liquid chromatography-tandem mass spectrometry (Online 2D SPE-HPLC-MS/MS) method was developed. Sidestream cigarette smoke was collected in a pre-treated (with ascorbic acid) fishtail chimney and a pre-treated (with ascorbic acid) Cambridge filter pad. The Cambridge filter pad post smoking was placed in an Erlenmeyer flask, and TSNAs adsorbed on fishtail chimney was rinsed by ammonium acetate solution. After extraction, the solution was filtered and cleaned by online 2D SPE, and analyzed by LC-MS/MS. The results showed that:1) Comparing with online SPE on a single cation exchange cartridge or a single reversed phase cartridge, 2D SPE gave the best overall cleanup, removed interfering substances more effectively, and minimized any sample matrix effect. 2) The optimized 2D SPE conditions were:sample loading 0.8 mL of 2% FA at a flow rate of 1.0 mL/min, sample transferring 0.8 mL of 1% NH₄OH at a flow rate of 0.8 mL/min, washing liquid for PRS cartridge 2% FA containing 15% MeOH, washing liquid for C₁₈ HD cartridge 1% NH₄OH, and elution time 1.5 min. 3) The detection limits for NNN, NAB, NAT and NNK were 0.006, 0.002, 0.004 and 0.003 ng/mL, respectively. The recoveries ranged from 93.4% to 107.3% and the relative standard deviations were less than 8%.This new approach is sensitive, accurate and suitable for the determination of NNN, NAB, NAT and NNK in sidestream cigarette smoke.