DNA crosslinking agents make up a broad class of chemotherapy agents that target rapidly dividing cancer cells by disrupting DNA synthesis. These drugs differ widely in both chemical structure and biological effect. In cells, crosslinking agents can form multiple types of DNA lesions with varying efficiencies. Inter-strand crosslinks (ICLs) are considered to be the most cytotoxic lesion, creating a covalent roadblock to replication and transcription. Despite over 50 years in the clinic, the use of crosslinking agents that specialize in the formation of ICLs remains limited, largely due to high toxicity in patients. Current ICL-based therapeutics have focused on late-stage and drug-resistant tumors, or localized treatments that limit exposure. In this article, we review the development of clinical crosslinking agents, our understanding of how cells respond to different lesions, and the potential to improve ICL-based chemotherapeutics in the future.