Introduction: Ineffective response of conventional therapies for the treatment of cancer has dictated the search for new treatment strategies.Emerging mode of cancer therapy likeoncolytic virotherapy employs oncolytic viruses (OVs) like Newcastle disease virus which can selectively kill cancer cells through direct lysis and can also trigger a potent anti-tumor immune response. Aim: To study the immunostimulatory effect of NDV9B on cancer cell lines MCF 7, MDA MB-231, A549, PC3, and normal HEK-293 cell line. Methodology: UV inactivated NDV9B HAU 10 and 20was used to infect human PBMC. Post infection the stimulation of cytokine IFN-α, IFN-γ and TRAIL was quantified using ELISA. These activated PBMC was co-cultured with the cancer and control normal cell lines to check tumor neutralization activity. Results: UV inactivated NDV9B triggered cytokines secretion in PBMCs. The concentration of cytokine IFN-α, IFNγ, TRAIL/ TNFSF10 was found to be 239.57 pg/mL, 14.68 pg/mL, 61.17 pg/mL respectively infected with 20 HAU. Activated PBMCs when co cultured with cancer cells showed maximum cytotoxicity of 11.75 % in MDA MB231 cancer cell line. Conclusion: NDV9B was found to stimulate cytokines in PBMCs and these PBMCs successfully brought about low level of cytopathic effect in MDA MB231.