O6-methylguanine-DNA　methyltransferase　(MGMT)　constitutes　an　important　cellular　mechanism　for　repairing　potentially　cytotoxic　DNA　damage　induced　by　guanine　O6-alkylating　agents　and　can　render　cells　highly　resistant　to　certain　cancer　chemotherapeutic　drugs.　A　wide　variety　of　potential　MGMT　inactivators　have　been　designed　and　synthesized　for　the　purpose　of　overcoming　MGMT-mediated　tumor　resistance.　We　determined　the　inactivation　potency　of　these　compounds　against　human　recombinant　MGMT　using　[3H]-methylated-DNA-based　MGMT　inactivation　assays　and　calculated　the　IC50　values.　Using　the　results　of　370　compounds,　we　performed　quantitative　structure–activity　relationship　(QSAR)　modeling　to　identify　the　correlation　between　the　chemical　structure　and　MGMT-inactivating　ability.　Modeling　was　based　on　subdividing　the　sorted　pIC50　values　or　on　chemical　structures　or　was　random.　A　total　of　nine　molecular　descriptors　were　presented　in　the　model　equation,　in　which　the　mechanistic　interpretation　indicated　that　the　status　of　nitrogen　atoms,　aliphatic　primary　amino　groups,　the　presence　of　O-S　at　topological　distance　3,　the　presence　of　Al-O-Ar/Ar-O-Ar/R.O.R/R-O-C=X,　the　ionization　potential　and　hydrogen　bond　donors　are　the　main　factors　responsible　for　inactivation　ability.　The　final　model　was　of　high　internal　robustness,　goodness　of　fit　and　prediction　ability　(R2pr　=　0.7474,　Q2Fn　=　0.7375–0.7437,　CCCpr　=　0.8530).　After　the　best　splitting　model　was　decided,　we　established　the　full　model　based　on　the　entire　set　of　compounds　using　the　same　descriptor　combination.　We　also　used　a　similarity-based　read-across　technique　to　further　improve　the　external　predictive　ability　of　the　model　(R2pr　=　0.7528,　Q2Fn　=　0.7387–0.7449,　CCCpr　=　0.8560).　The　prediction　quality　of　66　true　external　compounds　was　checked　using　the　“Prediction　Reliability　Indicator”　tool.　In　summary,　we　defined　key　structural　features　associated　with　MGMT　inactivation,　thus　allowing　for　the　design　of　MGMT　inactivators　that　might　improve　clinical　outcomes　in　cancer　treatment.　©　2023　by　the　authors.