Genetic alterations in members of the nucleotide excision repair (NER) pathway are present in a wide spectrum of cancers, but specific treatment options for this patient population are scarce. Here, we show occurrence of putative damaging germline and somatic alterations in NER genes in up to 10% of patients within certain cancer types across a large set of cancers and explored the potential therapeutic role of irofulven for patients with hypomorphic mutations in nucleotide excision repair genes. Gene-edited isogenic pairs of wildtype and mutant ERCC2 or ERCC3 cell lines were used to assess response to irofulven. Both in vitro and in vivo studies showed significantly enhanced irofulven sensitivity in cells harboring specific clinically observed heterozygous mutations in ERCC2 or ERCC3. Sensitivity of NER mutants to irofulven was greater than to the current standard of care agent cisplatin. Hypomorphic ERCC2/3 mutant cells had an impaired ability to repair irofulven induced DNA damage. Transcriptomic profiling of the tumor tissues suggested co-dependencies between DNA repair pathways, indicating a potential benefit of combination therapies, which were confirmed by in vitro studies. The present study provides a molecularly targeted, pre-clinical approach to cancers with mutations in nucleotide excision repair pathway genes, demonstrating preferential sensitivity to the drug irofulven alone, or in combination with other agents. Overall design: Analysis of mRNA expression in xenografts derived from either wildtype human mammary epithelial (HMLE) cells or HMLE cells gene-edited to harbor a heterozygous mutation in ERCC3 (p.R109X). Tumors from eight vehicle treated mice each and tumors from seven mice each treated with irofulven were used for analysis.