Olaparib is selected based on the presence of BRCA mutations in patient populations; however, further investigation is still required regarding its effect on restoring homologous recombination (HR) through the inactivation of non-homologous end joining (NHEJ). Therefore, identifying regulators of NHEJ could increase the sensitivity of cancer cells to Olaparib by inhibiting DNA damage repair is a major focus of current research. Loss of DNA-dependent protein kinase (DNA-PK), which is a major components of NHEJ, compromises DNA damage repair, and the resulting increase in DNA damage burden may heighten reliance on poly (ADP-ribose) polymerase (PARP)-dependent DNA repair in cancer cells, rendering them more susceptible to PARP inhibitor therapy. However, DNA-PK alone is not sufficient to enhance the effectiveness of Olaparib, so various adjuvant and combination therapies are being explored. We classified colorectal cancer (CRC) cells based on their sensitivity to Olaparib and found that they were categorized according to TP53 status. Here, we examine the role of DNA-PK in the response to Olaparib, emphasizing its relationship with TP53 status. Our findings indicate that the inhibition of DNA-PK enhances sensitivity to Olaparib and induces phosphorylation of p53 exclusively in cells with TP53 wild-type (WT). Furthermore, using CRC patient-derived cells (PDC) and patient-derived xenograft (PDX) model, we show that the sensitivity of Olaparib is determined TP53 and DNA-PK genotypes. These findings highlight TP53 and DNA-PK as potential predictive biomarkers for optimizing PARP inhibitor-based therapy in CRC.