Many tumors contain mutations that affect the DNA Damage Response (DDR) and genome stability. These mutations differentiate tumour cells from normal surrounding tissue and could be targeted by therapeutics that are based on negative genetic interactions with these mutations. While some genetic interactions result in synthetic lethality, other genetic interactions may increase the sensitivity of tumor cells to DNA damaging therapeutic agents, resulting in a class of conditional synthetic lethality we call Synthetic Cytotoxicity (SC). To identify SC interactions, we are screening yeast DDR mutants for SC interactions with several different DNA damaging agents using both a genome-wide array of non-essential mutants and a curated mini-array of mutants enriched for DDR genes. To date, we have screened mutations affecting the DNA damage checkpoint Tel1 (the yeast ATM ortholog) and the four core components of the non-homologous end joining (NHEJ) machinery: Yku70/Yku80 (DNA end–binding Ku heterodimer) and Dnl4/Lif1 (DNA ligase IV) for SC with four different DNA damaging agents: Bleomycin (radio-mimetic), Camptothecin (TopI inhibitor), Cisplatin (interstrand crosslinker), and MMS (alkylating agent). We characterized and investigated the biology of several strong SC interactions and tested for evolutionary conservation in C. elegans. Our SC interaction network of DDR pathways will help interpret the complex networks that maintain genome stability after exposure to DNA damaging therapeutic agents, and will provide a valuable resource for the rational design of personalized therapy based on tumour genotypes.