Non-small cell lung cancer has a devastating prognosis, and markers enabling a precise prediction of therapy response have long remained scarce. Better treatment monitoring would allow an individual's more effective patient adjusted therapy with lesser side effects and good clinical outcomes. In the present study, we monitored the serum cytochrome c levels pre- and post-chemotherapy of non-small cell lung cancer patients. Using highly sensitive enzyme-linked immunosorbent assay, we evaluated cytochrome c levels in serum of 100 non-small cell lung cancer and 100 healthy controls. We observed about threefold lower serum cytochrome c level in newly diagnosed non-small cell lung cancer patients than healthy individuals. Patients in advanced stages and grade 3 histological differentiation showed significantly low level of serum cytochrome c, and the lower levels were associated with worse survival outcome of non-small cell lung cancer patients. In addition, serum cytochrome c level was observed to be more than 13-fold higher after first cycle of conventional chemotherapy, wherein patients with higher level of serum cytochrome c before any therapy showed better response to chemotherapy in terms of significantly higher level of serum cytochrome c after first cycle of chemotherapy than patients with low level of serum cytochrome c at the time of diagnosis. Detection of serum cytochrome c levels at the time of diagnosis may be useful in suggesting disease severity and prognosis of the non-small cell lung cancer patients. Monitoring of serum cytochrome c might also serve as a sensitive apoptotic marker in vivo reflecting chemotherapy-induced cell death burden in patients with non-small cell lung cancer.