Abstract: Sulfur mustard (2, 2’-dichloroethyl-sulfide, HD) is an alkylating agent and a powerful vesicant, which was widely imposed and caused the bulk of injuries and deaths in World War Ⅰ and Iran-Iraq War. Since World War Ⅱ, many casualties have been caused by the chemical weapons containing HD abandoned by Japanese in China. It still represents potential threat to military and civilian population. It’s very important to study reliable bioanalytical methods for detecting HD exposure. HD reacts with histidine residues of hemoglobin producing the HD adduct which has been used as a biomarker for retrospective detection of HD exposure because of its long half-life (about 120 days). A novel method based on the HD-hemoglobin adduct for qualitative and quantitative detection of trace HD exposure was developed. The HD-hemoglobin was isolated from blood through centrifugation and hemolyzation in water. Then the heme was removed with acidic acetone to obtain the HD-globin. HD-globin adduct was digested with pronase, purified by PPL solid phase extraction following benzyl chloroformate (Cbz-Cl) derivatization, and analyzed with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Upon optimization, the digestion was performed at 55 ℃ for 10.0 h. The PPL loading samples was washed with 3.0 mL 5% methanol solution and eluted with 1.0 mL 50% methanol solution. The derivatization time and temperature were 30 min and 4 ℃, respectively. With 2-chloroethyl ethyl sulfide spiked hemoglobin solution serving as internal standard (ISTD), the method was found to be linear between 10.0 and 1 000 μg/L HD exposure (R²>0.997) with precision of ≤12.4% and accuracy ranged between 89.8% and 113%. In spite of a shorter sample treatment time (compared with the acid hydrolysis, which needed reflux in hydrochloric acid at 110 ℃ for 24 h), the limit of detection (LOD) was 10.0 μg/L (S/N>5) which was 150 times lower than that by the acid hydrolysis in a previous article. Furthermore, two transitions were more beneficial to retrospective detection for trace HD exposure than only one transition used in the previous article. This method showed great specificity with absent interference, stable ratio of confirmation transition, quantitative transition, mild sample preparation conditions and simplified operations. This study provides a method for the retrospective detection of trace-HD exposure, which is beneficial to the official Organization for the Prohibition of Chemical Weapons (OPCW) proficiency tests. Furthermore, this method can provide evidence for allegations of the use of HD, treatments of the persons exposed to HD and the study of toxication mechanism.