Human papilloma virus (HPV)-associated oropharyngeal squamous cell carcinoma (HPV-OPSCC) has been recognized to have an excellent response to treatment and has a distinct pathologic staging. For this reason, HPV testing is recommended in cytology specimens of metastatic OPSCC, although the guidelines for testing are not clearly defined. The aim of the current study was to establish a threshold for interpretation of p16 in aspirates from metastatic OPSCC.Cases diagnosed as metastatic SCC by cytology in neck lymph nodes with concurrent p16 on cytology and on paired surgical specimen or an in situ hybridization (ISH) for HPV were included in the study. Stain intensity and percentage positive cells for p16 was compared with p16 on paired surgical pathology specimens and/or ISH RNA for HPV on cytology specimens.Of the 52 cases diagnosed as metastatic SCC on neck aspirates, paired surgical pathology specimens and/or ISH HPV was available in 17 cases. A p16 expression in ≥10%-15% cells resulted in a sensitivity and negative predictive value of 66% and 37%, respectively. However, when even minimal expression in tumor fragments is considered positive, the negative predictive value increases to 100%.We recommend that even minimal nuclear expression for p16 in viable tumor fragments must be considered as positive in cytology specimens. Expression limited to only background single tumor cells or in a necrotic specimen must be interpreted with caution.