Inhibition of the DNA damage response is an emerging strategy to treat cancer. Understanding how DNA damage response inhibitors cause cytotoxicity in cancer cells is crucial to their further clinical development. This review focuses on three different mechanisms of cell killing by checkpoint kinase I inhibitors (CHK1i). DNA damage induced by chemotherapy drugs, such as topoisomerase I inhibitors, results in S and G2 phase arrest. Addition of CHK1i promotes cell cycle progression before repair is completed resulting in mitotic catastrophe. Ribonucleotide reductase inhibitors such as gemcitabine also arrest cells in S phase by preventing dNTP synthesis. Addition of CHK1i re-activates the DNA helicase to unwind DNA, but in the absence of dNTPs, this leads to excessive single-strand DNA that exceeds the protective capacity of the single-strand-binding protein RPA. Unprotected DNA is subjected to nuclease cleavage, resulting in replication catastrophe. CHK1i alone also kills a subset of cell lines through MRE11 and MUS81-mediated DNA cleavage in S phase cells. The choice of mechanism depends on the activation state of CDK2. Low level activation of CDK2 mediates helicase activation, cell cycle progression, and both replication and mitotic catastrophe. In contrast, high CDK2 activity is required for sensitivity to CHK1i as monotherapy. This high CDK2 activity threshold usually occurs late in the cell cycle to prepare for mitosis, but in CHK1i-sensitive cells, high activity can be attained in early S phase, resulting in DNA cleavage and cell death. This sensitivity to CHK1i has previously been associated with endogenous replication stress, but the dependence on high CDK2 activity, as well as MRE11, contradicts this hypothesis. The major unresolved question is why some cell lines fail to restrain their high CDK2 activity and hence succumb to CHK1i in S phase. Resolving this question will facilitate stratification of patients for treatment with CHK1i as monotherapy.