The　repair　of　DNA　mediated　by　O-6-alkylguanine-DNA　alkyltransferase　(AGT)　provides　protection　against　DNA　damage　from　endogenous　or　exogenous　alkylation　of　the　O-6　position　of　guanine.　However,　this　repair　acts　as　a　double-edged　sword　in　cancer　treatment,　as　it　not　only　protects　normal　cells　from　chemotherapy　associated　toxicities,　but　also　results　in　cancer　cell　resistance　to　guanine　O-6-alkylating　antitumour　agents.　Thus,　AGT　plays　an　important　role　in　predicting　the　individual　susceptibility　to　guanine　O-6-alkylating　carcinogens　and　chemotherapies.　Accordingly,　it　is　necessary　to　establish　a　quantitative　method　for　determining　AGT　activity　with　high　accuracy,　sensitivity　and　practicality.　Here,　we　describe　a　novel　non-radioactive　method　for　measuring　AGT　activity　using　stable　isotope　dilution　high-performance　liquid　chromatography　electrospray　ionization　tandem　mass　spectrometry　(HPLC-ESI-MS/MS).　This　method　is　based　on　the　irreversibility　of　the　removal　of　the　O-6-alkyl　group　from　guanine　by　AGT　and　on　the　high　affinity　of　O-6-benzylguanine　(O-6-BG)　as　an　AGT　substrate.　HPLC-ESI-MS/MS　was　used　to　measure　the　AGT　activities　in　cell　protein　extracts　from　eight　tumour　lines,　demonstrating　that　AGT　activity　was　quite　variable　among　different　cell　lines,　ranging　from　nondetectable　to　1021　fmol/mg　protein.　The　experiments　performed　in　intact　tumour　cells　yielded　similar　results　but　exhibited　slightly　higher　activities　than　those　observed　in　cell　protein　extracts.　The　accuracy　of　this　method　was　confirmed　by　an　examination　of　AGT　expression　levels　using　western　blotting　analysis.　To　our　knowledge,　this　method　is　the　first　mass　spectrometry-based　AGT　activity　assay,　and　will　likely　provide　assistance　in　the　screening　of　cancer　risk　or　the　application　of　chemotherapies.　(C)　2016　Elsevier　B.V.　All　rights　reserved.