Cigarette is one of the factors that increases the numbers of human deaths because it contains many harmful substances, such as benzo[a]pyren (B[a]P) and heavy metal Cr(VI). One of the consequences caused by both substances is an oxidative DNA damage and DNA adducts 8-hydroxy-2'-deoxyguanosine (8-OHdG) production as a biomarker of cancer risk. In this research, in vitro assay of benzo[a]pyrene with Fenton-like reagent was conducted in several pHs (7.4 and 8.4), temperatures (37°C and 60°C), and incubation times (5 and 7 hours). The 8-OHdG DNA adducts was analyzed using a reverse phase HPLC chromatography with UV/vis detector at the wavelength of 254 nm. The result shows that the 8-OHdG concentration due to exposure of B[a]P in the presence of Fenton-like reagent has exceeded the minimum concentration of detection that was set as LOD value of 5.188 ppb. Based on the data result, the increasing time and temperature incubation have a synergistic effect of the 8-OHdG-concentration increase. The highest concentration of 8-OHdG formation was conducted in the condition of pH 7.4, temperature 60°C, and incubation time 12 h in the presence of B[a]P, Cr(VI), H2O2 and vitamin C as much as 15.13 ppb.