Oxidation of DNA due to exposure to reactive oxygen species (ROS) is a major source of DNA damage.ROS induced damage to DNA plays an important role in some diseases such as various cancers, aging andneurodegenerative diseases. The detection of DNA oxidation products plays a major role in assessing themutagenicity potential of specific exposure. The GC–MS/MS method was developed for the ultrasensi-tive determination of individual DNA damage products. The validation results revealed that the proposedmethod was reliable and sensitive. Multiple response surface methodology (MRSM) was used to opti-mize derivatization conditions of oxidatively DNA base damage products before GC–MS/MS analysis.The optimum derivatization conditions were determined as 40 min for derivatization time, 120◦C forderivatization temperature and 1.4 for BSTFA/pyridine ratio under nitrogen atmosphere. The effects ofthymol, carvacrol and thymoquinone as antioxidants were investigated on oxidative DNA damage. Thedetermination of the oxidatively induced DNA damage products was performed after adding DNA andantioxidants with different concentrations under oxidative stress. Eighteen DNA base damage productswere analyzed simultaneously using GC–MS/MS. This study showed a significant decrease in the amountof DNA base damage products when the antioxidants were present in the medium.