Mammalian checkpoint kinases 1 and 2 (Chk1 and Chk2) are essential kinases that are involved in cell cycle checkpoint control, and the abrogation of either has been proposed as one way to sensitize cancer cells to DNA-damaging agents. However, it remains unclear which kinase is the most therapeutically relevant target, and whether multiple kinases might need to be targeted to achieve the best efficacy because of their overlapping substrate spectra and redundant functions. To clarify this issue, we established asynchronous cell cycle arrest models to investigate the therapeutic outcomes of silencing Chk1 and Chk2 in the presence of irradiation or cisplatin. Our results showed that Chk1- and Chk2-targeting small interference RNAs (siRNAs) demonstrated synergistic effects when both siRNAs were used simultaneously. Interestingly, Chk1 and Chk2 co-expression occurred in similar to 90% of neoplastic tissues examined and showed no difference in neoplastic versus non-neoplastic tissues. Therefore, the selective targeting of Chk1 and Chk2 by oncolytic adenovirus mutants was chosen to treat resistant tumor xenograft mice, and the maximum antitunnoral efficacy was achieved with the combined co-abrogation of Chk1 and Chk2 in the presence of low-dose cisplatin. This work deepens our understanding of novel strategies that target checkpoint pathways and contributes to the development of novel, potent and safe checkpoint abrogators.