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Abstract Ionising radiation (IR) is widely used in cancer treatment, including for head and neck squamous cell carcinoma (HNSCC), where it induces significant DNA damage leading ultimately to tumour cell death. Among these lesions, DNA double strand breaks (DSBs) are the most threatening lesion to cell survival. The two main repair mechanisms that detect and repair DSBs are non-homologous end joining (NHEJ) and homologous recombination (HR). Among these pathways, the protein kinases ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and the DNA dependent protein kinase catalytic subunit (DNA-Pkcs) play key roles in the sensing of the DSB and subsequent coordination of the downstream repair events. Consequently, targeting these kinases with potent and specific inhibitors is considered an approach to enhance the radiosensitivity of tumour cells. Here, we have investigated the impact of inhibition of ATM, ATR and DNA-Pkcs on the survival and growth of six radioresistant HPV-negative HNSCC cell lines in combination with either X-ray irradiation or proton beam therapy, and confirmed the mechanistic pathway leading to cell radiosensitisation. Using inhibitors targeting ATM (AZD1390), ATR (AZD6738) and DNA-Pkcs (AZD7648), we observed that this led to significantly decreased clonogenic survival of HNSCC cell lines following both X-ray and proton irradiation. Radiosensitisation of HNSCC cells grown as 3D spheroids was also observed, particularly following ATM and DNA-Pkcs inhibition. We confirmed that the inhibitors in combination with X-rays and protons led to DSB persistence, and increased micronuclei formation. Cumulatively, our data suggest that targeting DSB repair, particularly via ATM and DNA-Pkcs inhibition, can exacerbate the impact of ionising radiation in sensitising HNSCC cell models.