Purpose: Squamous cell carcinoma metastasis of the head and neck with unknown primary tumor (CUP) comprises a diagnostic challenge. Human papillomavirus (HPV) testing on cytologic specimens is gaining increasing focus as this may facilitate an early diagnosis of HPV-induced oropharyngeal carcinoma. This study aimed to prospectively assess PCR-based HPV-DNA testing on FNA smears in a clinical setting. Methods: Patients referred to a tertiary Head and Neck Cancer Center with suspected CUP were included from November 2016 to November 2018. Scraped cell material from FNA smears was analyzed for HPV-DNA with PCR using general primers (GP5 + /GP6 +) and correlated with the origin and histology of the primary tumor (oropharynx vs. outside oropharynx or benign tumor). The turn-around time reflecting the workflow for HPV-DNA testing by PCR was also calculated. Results: A total of 93 patients were enrolled in the study. The sensitivity and specificity were 86.7% [95% CI 75.4–94.1%] and 92.0% [95% CI 74.0–99.0%], and the positive and negative predictive values were 96.3% [95% CI 87.3–99.0%] and 74.2% [95% CI 59.9–84.7%], respectively. The turn-around time for HPV testing was a mean four calendar days. Conclusion: HPV-DNA testing on FNA smears can be performed within a reasonable timeframe and can guide for the detection of an HPV-positive oropharyngeal primary tumor in the clinical setting for patients presenting with CUP of the head and neck.