Previous studies have described that tumor organoids can capture the diversity of defined human carcinoma types. Here, we describe conditions for long-term culture of human mucosal organoids. Using this protocol, a panel of 31 head and neck squamous cell carcinoma (HNSCC)-derived organoid lines was established. This panel recapitulates genetic and molecular characteristics previously described for HNSCC. Organoids retain their tumorigenic potential upon xenotransplantation. We observe differential responses to a panel of drugs including cisplatin, carboplatin, cetuximab and radiotherapy in vitro. Drug screens reveals selective sensitivity to targeted drugs that are not normally used in the treatment of HNSCC patients. These observations may inspire a personalized approach to the management of HNSCC and expand the repertoire of HNSCC drugs.