The incidence of malignant melanoma is growing rapidly worldwide and while there are now a number of targeted therapies for treatment of metastatic disease, rapid development of resistance and restricted expression of targets means there are still a significant proportion of patients without effective treatment options. Conventional chemotherapeutic drugs damage DNA and create cellular stresses in the proliferating cells including the tumor cells in the patients and induce the cell cycle checkpoint arrest. Thus, standard chemotherapeutic treatments need to use high doses of drugs and produce low response rates with adverse side effects. Chk1 inhibitors are being investigated for their abilities to abrogate the cell cycle checkpoint arrest and, as a result, enhance the effects of conventional chemotherapy, but their activity as single agents is relatively unknown. We have investigated the activity of Chk1 inhibitor GNE323 in 40 different melanoma cell lines and normal cells in vitro. This single-agent treatment with GNE323 produces cytotoxic effects in low nanomolar range in a subset of melanoma cell lines with high levels of replicative stress, but only cytostatic effects in normal cells at the higher drug concentration. Our results also show that this cytotoxic effect in cell lines most sensitive to Chk1 inhibition is related to cells undergoing apoptosis either prior to or during an aberrant mitosis due to inhibition of S phase Chk1. This sensitivity suggests that Chk1 play an integral part in these drug-sensitive melanoma cells’ adaptation to replicative stress and as the sensitivity to Chk1 inhibitor is independent of the currently targeted melanoma mutations, Chk1 inhibitors may be useful as a single-agent therapy for the currently untreated patient population or in patients with developed resistance to MAPK targeted agents.