NAD⁺ is an important coenzyme involved in various metabolic processes. NAD⁺ is also used as a substrate for poly-ADP-ribose polymerase (PARP). DNA single strand break (SSB) induces auto-ADP-ribosylation of PARP and recruits DNA repair complex. It is known that oxidative stress, such as hydrogen peroxide (H₂O₂), induces SSB DNA damage and deplete NAD⁺ via PARP-mediated poly ADP-ribosylation. The treatment with low dose of H₂O₂ induces NAD⁺ depletion, but cannot kill the cells. Therefore, we pursuit the method to induce the synthetic lethality with low dose of H₂O₂. We induced DNA damage in A549 cells by adding low dose of H₂O₂. We found that NAD⁺ level significantly declined at 1 hour after H₂O₂ treatment, but recovered to normal level thought the resynthesis of NAD⁺ at 24 hours later. Next, we investigated how NAD⁺ is re-synthesized after the low dose of H₂O₂ treatment. In particular, the source of ribose moiety of NAD⁺ was unknown. Using stable isotope labeled glucose, we identified that phosphoribosyl pyrophosphate (PRPP), the source of ribose moiety of NAD⁺, comes from the glucose but not the ADP-ribose, a degradation product of auto-ADP-ribosylated PARP. Next, we examined the effect of these combination to induce cell death in cancer cells. Single treatment of H₂O₂ or glucose depletion did not induce cell death, but the combination of the low dose of H₂O₂ treatment and glucose depletion could induce synthetic lethality in A549 cells. These results demonstrate that the combination of oxidative stress and NAD⁺ synthesis inhibition can be an optimal therapeutic option to kill the cancer cells with less invasiveness.