Radiotherapy is one of the promising, effective and common modalities for the treatment of cancer in spite of the potentially severe side effects. Irradiation of cancer cells induces double stranded DNA breakage (DSB) which is a critical lesion that is repaired by DSB repair pathways. Activation of DSB repair is one of the main causes of radiation resistance and failure of treatment. DSBs are repaired by various pathways such as non-homologous end joining (NHEJ) or homologous recombination (HR). A major mechanism by which cancer cells become resistant to ionizing radiation (IR) and chemotherapy is by enhanced DSBs repair. DNA dependent protein kinase (DNA-PK) is an essential kinase family enzyme in the NHEJ pathway. Targeting DNA-PK using strategically selective inhibitors is a major interest in cancer therapeutics research. The purpose of this study is to determine the in vitro DNA-PK inhibitory effect of novel derivatives of substituted benzoxazines following irradiation. Various cell lines such as HeLa cervical cancer, HT 29 colorectal adenocarcinoma, A 549 lung carcinoma and WI38 normal lung fibroblasts were treated with selected compounds and irradiated with 1 to 8 Gy using a Varian linear accelerator. Various assays such as the clonogenic assay, ɣH2AX assay and cell cycle analysis were conducted to determine survival rate, quantify DSB’s and apoptosis respectively. We found a significant reduction in the survival rate in for HT 29 and A 549 cell lines after treating them concomitantly with both radiation and our compounds. The ɣH2AX phosphorylation indicating the presence of DSB was found to be prolonged significantly to 24 hours indicating the presence of unrepaired DSB’s. There was also significant increase in the number of cells in the Sub G1 phase of the cell cycle, indicating an increased number of cells in apoptosis. All our results reveal the effectiveness of selected, novel benzoxazines as promising radiation sensitizing agents.