CHK1 inhibitors are being investigated as chemosensitising agents with agents that increase replication stress. Here we have investigated the molecular basis of sensitivity to CHK1 inhibitors as single agents in melanoma and as combinations with subclinical doses of hydroxyurea in melanoma and non-small cell lung cancer (NSCLC). We have found that sensitivity in vitro and in vivo to single agent CHK1 inhibitor is associated with defective S phase cell cycle checkpoint response. Loss of checkpoint response by over-expressing components of the checkpoint or inhibition of Wee1, convert CHK1 inhibitor insensitive cells to sensitive, although the sensitization with We1 inhibitor is surprisingly dependent the level of endogenous replication stress. Similarly, depletion of CDC25A reduces CHK1 inhibitor sensitivity in sensitive lines. In the CHK1 inhibitor sensitive cells, cell death was triggered without to entry into mitosis. Sensitivity t CHK1 inhibitor correlated with the presence of high level endogenous replication stress marked by phosphor-RPA foci. We have found that about 20% of melanomas are sensitive to CHK1 inhibitor as a single agent, but we show that >70% of melanomas and NSCLCs are sensitive to combination with subclinical doses of hydroxyurea. The mechanism of cell death in the combination appears identical to CHK1 inhibitor alone in inhibitor-sensitive melanomas, with cells dying without entering mitosis, and death apparently dependent on increased NOXA expression. In half the cell melanomas tested, the combination produced a complete loss of viability in the other half, the viable population after treatment was incapable of further proliferation in the absence in drug. The sensitivity to the combination is independent of other known risk factors, suggesting a significant proportion of melanoma and lung cancer patients could benefit from treatment with this drug combination.