Genomic instability, a hallmark of almost all human cancers, drives both carcinogenesis and resistance to therapeutic interventions. Pivotal to the ability of a cell to maintain genome integrity are mechanisms that signal and repair deoxyribonucleic acid (DNA) double-strand breaks (DSBs), one of the most deleterious lesions induced by ionising radiation and various DNA-damaging chemicals. On the other hand, many current therapeutic regimens that effectively kill cancer cells are based on the induction of excessive DSBs. However, these drugs often lack selectivity for tumour cells, which results in severe side effects for the patients, thus compromising their therapeutic potential. Therefore, the development of novel tumour-specific treatment strategies is required. Unlike normal cells, however, cancer cells are often characterised by abnormalities in the DNA damage response including defects in cell cycle checkpoints and/or DNA repair, rendering them particularly sensitive to the induction of DSBs. Therefore, new anticancer agents designed to exploit these vulnerabilities are becoming promising drugs for enhancing the specificity and efficacy of future cancer therapies. Here, we summarise the latest preclinical and clinical developments in cancer therapy based on the current knowledge of DSB signalling and repair, with a special focus on the combination of small molecule inhibitors with synthetic lethality approaches.