Novel targeted agents to inhibit DNA repair pathways to sensitize tumors to irradiation (IR) are being investigated as an alternative to chemoradiation for locally advanced human papilloma virus negative (HPV-negative) head and neck squamous cell carcinoma (HNSCC). Two well-characterized targets that, when inhibited, exhibit potent IR sensitization are PARP1 and DNA-PKcs. However, their cooperation in sensitizing HPV-negative HNSCC to IR remains to be explored given that PARP1 and DNA-PkCS bind to unresected stalled DNA replication forks and cooperate to recruit XRCC1 to facilitate double-strand break repair. Here, we show that the combination of the DNA-PK inhibitor NU7441 and the PARP inhibitor olaparib significantly decrease proliferation (61–78%) compared to no reduction with either agent alone (p < 0.001) in both SCC1 and SCC6 cell lines. Adding IR to the combination further decreased cell proliferation (91–92%, p < 0.001) in SCC1 and SCC6. Similar results were observed using long-term colony formation assays [dose enhancement ratio (DER) 2.3–3.2 at 4Gy, p < 0.05]. Reduced cell survival was attributed to increased apoptosis and G2/M cell cycle arrest. Kinomic analysis using tyrosine (PTK) and serine/threonine (STK) arrays reveals that combination treatment results in the most potent inhibition of kinases involved in the CDK and ERK pathways compared to either agent alone. In vivo, a significant delay of tumor growth was observed in UM-SCC1 xenografts receiving IR with olaparib and/or NU7441, which was similar to the cisplatin-IR group. Both regimens were less toxic than cisplatin-IR as assessed by loss of mouse body weight. Taken together, these results demonstrate that the combination of NU7441 and olaparib with IR enhances HPV-negative HNSCC inhibition in both cell culture and in mice, suggesting a potential innovative combination for effectively treating patients with HPV-negative HNSCC.