Abstract: In order to evaluate toxicity of crotonaldehyde in cigarette smoke, calf thymus DNA damage caused by crotonaldehyde exposure was investigated at different concentrations of crotonaldehyde. After exposure, calf thymus DNA samples were firstly hydrolyzed by enzymes, then purified by solid phase extraction (SPE), concentrated and re-dissolved subsequently. Finally, the DNA adducts of S-α-CH₃-γ-OH-1, N₂-propanodeoxyguanosine (CdG-1) and R-α-CH₃-γ-OH-1, N₂-propano-deoxyguanosine (CdG-2) induced by crotonaldehyde exposure were determined by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/ MS). The relationships between CdG adducts and crotonaldehyde exposure dose were established. The results indicated that: 1) Good separation efficiency and short analysis time were achieved by using a C₁₈ column under gradient elution conditions. 2) The calibration curves presented good linearity (R₂>0.999) with the detection limits of CdG-1 and CdG-2 at 0.006 and 0.005 ng/mL, respectively. The recoveries ranged from 83.3% to 101.0% with the relative standard deviations (RSDs) less than 7.0%. 3) The content of CdG adducts in calf thymus DNA increased with the increase of crotonaldehyde exposure dose, namely, dose-response relationship between them. The developed method is simple and accurate, thus can be used as an effective tool for evaluating the DNA damage caused by crotonaldehyde exposure.