O6-methylguanine (O6meG) is one of the most premutagenic, precarcinogenic, and precytotoxic DNA lesions formed by alkylating agents. Repair of this DNA damage is achieved by the protein MGMT, which transfers the alkyl groups from the O6 position of guanine to a cysteine residue in its active center. Because O6meG repair by MGMT is a stoichiometric reaction that irreversibly inactivates MGMT, which is subsequently degraded, the repair capacity of O6meG lesions is dependent on existing active MGMT molecules. In the absence of active MGMT, O6meG is not repaired, and during replication, O6meG:T mispairs are formed. The MMR system recognizes these mispairs and introduces a gap into the strand. If O6meG remains in one of the template strands the futile MMR repair process will be repeated, generating more strand breaks (SBs). The toxicity of O6meG is, therefore, dependent on MMR and DNA SB induction of cell death. MGMT, on the other hand, protects against O6meG toxicity by removing the methyl residue from the guanine. Although removal of O6meG makes MGMT an important anticarcinogenic mechanism of DNA repair, its activity significantly decreases the efficacy of cancer chemotherapeutic drugs that aim at achieving cell death through the action of the MMR system on unrepaired O6meG lesions. Here, we report on a modification of the comet assay (CoMeth) that allows the qualitative assessment of O6meG lesions after their conversion to strand breaks in proliferating MMR-proficient cells after MGMT inhibition. This functional assay allows the testing of compounds with effects on O6meG levels, as well as on MGMT or MMR activity, in a proliferating cell system. The expression of MGMT and MMR genes is often altered by promoter methylation, and new epigenetically active compounds are being designed to increase chemotherapeutic efficacy. The CoMeth assay allows the testing of compounds with effects on O6meG, MGMT, or MMR activity. This proliferating cell system complements other methodologies that look at effects on these parameters individually through analytical chemistry or in vitro assays with recombinant proteins.