8-Hydroxy-2′-deoxyguanosine (8-OHdG) is the most common biomarker of oxidative DNA damage, it is formed by chemical carcinogens and can be measured in any species. Perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA) are suspected genotoxic carcinogens through induction of reactive oxygen species that are responsible for oxidative DNA damage. This study was conducted to investigate the in vitro genotoxicity of PFOA and PFNA in human lymphoblastoid (TK6) cell line. TK6 cells were exposed to PFOA at 0, 125, 250, and 500 ppm and PFNA at 125 and 250 ppm for 2 h. Single cell gel electrophoresis (comet assay) was used to measure DNA damage; at least 50 cells per sample were analyzed using comet Assay Software Project (CASP). 8-OHdG was measured in DNA of exposed cells using high-performance liquid chromatography (HPLC)-mass spectrometry (MS)/MS. Results showed that both PFOA and PFNA induced DNA damage indicated by increased tail length (DNA migration). The level of 8-OHdG was increased in a dose-dependent manner in both PFOA and PFNA exposure. We concluded that PFOA and PFNA induced DNA damage and the biomarker of oxidative DNA damage (8-OHdG) could be measured by HPLC-MS/MS. In addition, PFNA produced high level of 8-OHdG at concentrations lower than PFOA, this may indicate that PFNA is more potent genotoxicant for TK6 cells than PFOA. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 192–200, 2016.