Non-homologous end joining (NHEJ) is a major DNA double strand breaks (DSBs) repair pathway that maintains genome integrity. However, this pathway may reduce radiotherapy efficacy by repairing DSBs on cancer cells. This research reported a computer-aided drug design (CADD) method to identify novel inhibitors from traditional Chinese medicine (TCM) that disrupt NHEJ. We aim to inhibit Ku86, the initiator of NHEJ. By integrating binding energy evaluation and molecular dynamics simulation methods, we reported glycyrrhizic acid, macedonoside C, lithospermic acid, and salvianolic acid B as potential Ku86 inhibitors. All four TCM compounds show low binding energy and stable binding poses to Ku86. The carboxyl groups on a ligand are the major binding region by forming salt bridges at Ku86 binding sites. Additional features were defined by a carbonyl group or a dihydroxyphenyl group that form additional hydrogen bond or pi-cation respectively with the ligand binding site on Ku86. These features strengthen the binding affinity between Ku86 and the potential TCM ligand. We reported all four TCM compounds are potential Ku86 inhibitors and may be used to enhance radiotherapy for cancer treatment.