Abstract: 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 O6-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.