Recent studies have revealed an important and intriguing role for human papillomavirus (HPV) in head and neck squamous cell carcinoma (HNSCC). There are indications that the prevalence of HPV-positive HNSCC has recently increased, and genetic analyses point to a completely distinct class of HNSCCs. Most studies suggest that patients with this type of tumour have a better prognosis and some argue that an adjusted therapeutic approach is needed. One crucial point in the research of HNSCC-HPV involvement has often been neglected, which is the lack of a standardised assay to detect HPV. This has resulted in a considerable variation in the frequency of HPV-positive tumours between studies reported thus far. Especially for PCR-based tests, the risk exists that the assay is too sensitive and detects virus without implying a causal involvement in HNSCC. A reliable algorithm to detect a clinically relevant HPV infection in formalin-fixed paraffin embedded tissue has recently become available. Here, we address important biological and analytical aspects of HPV involved in the development of HNSCC and it is emphasised that a standardised HPV assay is a prerequisite for assessing the clinical importance of a HPV infection in HNSCC.