Resistance against common treatment regimens like chemotherapy is a major concern in current cancer research and clinical disease management. Moreover, in vitro cell culture models cannot always adequately represent the tumor situation in vivo, hampering the analysis of the underlying mechanisms. Efforts have been made to establish three-dimensional (3D) cancer cell models, such as spheroids or organoids, to mimick the in vivo situation of the tumor more closely. Here, we describe the establishment of a 3D spheroid model of head and neck squamous cell carcinoma (HNSCC), allowing for the study of chemoresistance. The analysis of relevant phenotypic readouts using a high-content screening platform enables the objective evaluation and monitoring of spheroids during chemotherapeutic treatment. This model also represents a starting point for the evaluation of a precision medicine-based management of HNSCC patients.