Head and neck cancers are characterized by both genetic and epigenetic aberrations. In treating head and neck cancers, ionizing radiation (IR) is an essential modality in either definitive or adjuvant setting. However, radiation-resistant head and neck cancers are not uncommon. The major biological determinator for IR resistance was previously considered at genetic level because DNA is the major target of irradiation damage. However, in head and neck cancers, recent evidence demonstrated epigenetic disturbance after IR, implicating its role in IR resistance. Hence, this chapter intends to establish an in vitro model for investigating DNA methylation changes in IR-resistant head and neck cancer cells. Bisulfite pyrosequencing is the main methodology it introduced.