Chloroethylnitrosoureas　(CENUs)　are　alkylating　agents,　which　are　used　widely　in　the　clinical　treatment　of　cancers.　CENUs　exhibit　anticancer　activity　by　inducing　DNA　interstrand　crosslinks　(ICLs),　mainly　dG-dC　crosslinks.　To　evaluate　the　anticancer　activity　and　drug　resistance　of　CENUs,　it　is　necessary　to　establish　a　highly　sensitive　method　for　the　quantitation　of　CENU-induced　DNA　ICLs　in　cells.　In　this　study,　NIH/3T3　fibroblasts　cells　and　L1210　leukemia　cells,　which　had　different　O6-alkylguanine-DNA　alkyltransferase　(AGT)　activity,　were　treated　with　Nimustine　(ACNU)　and　Carmustine　(BCNU).　The　levels　of　dG-dC　crosslinks　in　cells　were　determined　by　high-performance　liquid　chromatography-electrospray　ionization　tandem　mass　spectrometry　(HPLC-ESI-MS/MS).　The　limit　of　detection　(signal　to　noise　(S/N)=5)　and　limit　of　quantitation　(S/N=17)　achieve　to　2　fmol　and　8　fmol,　respectively.　The　recovery　is　range　from　92.5%　to　107.4%.　The　sensitivity　and　accuracy　of　the　quantitative　analysis　of　dG-dC　crosslinks　in　cells　are　satisfied.　The　results　indicate　that　the　dG-dC　level　induced　by　ACNU　is　higher　than　that　by　BCNU.　Moreover,　the　dG-dC　level　in　L1210　cells　is　obviously　higher　than　that　in　NIH/3T3　after　treated　at　the　same　drug　concentrations.　This　work　provides　a　reliable　method　for　evaluating　the　anticancer　activity　of　novel　CENU　alkylating　agents.　©,　2014,　Chinese　Society　for　Mass　Spectrometry.　All　right　reserved.