There have been hints that non-viral cancer antigens are differentially expressed in Human Papillomavirus (HPV) positive and HPV-negative head and neck squamous cell carcinoma (HNSCC). Antibody responses (AR) to cancer antigens may be used to indirectly determine cancer antigen expression in the tumor using a non-invasive and tissue-saving liquid biopsy. Here, we set out to characterize AR to a panel of non-viral cancer antigens in HPV-positive and HPV-negative HNSCC patients. A fluorescent microbead multiplex serology to 29 cancer antigens (16 cancer-testis antigens, 5 cancer-retina antigens, 8 oncogenes) and 29 HPV-antigens was performed in 382 HNSCC patients from five independent cohorts (153 HPV-positive, 209 HPV-negative). AR to any of the cancer antigens were found in 272/382 patients (72%). The ten most frequent AR were CT47, cTAGE5a, c-myc, LAGE-1, MAGE-A1, -A3, -A4, NY-ESO-1, SpanX-a1 and p53. AR to MAGE-A3, MAGE-A9 and p53 were found at significantly different prevalences by HPV-status. An analysis of AR mean fluorescent intensity values uncovered remarkably different AR clusters by HPV-status. To identify optimal antigen selections covering a maximum of patients with ≤10 AR, multiobjective optimization revealed distinct antigen selections by HPV-status. We identified that AR to non-viral antigens differ by HPV-status indicating differential antigen expression. Multiplex serology may be used to characterize antigen expression using serum or plasma as a tissue-sparing liquid biopsy. Cancer antigen panels should address the distinct antigen repertoire of HPV-positive and -negative HNSCC. This article is protected by copyright. All rights reserved.