Objective. To assess the feasibility of panitumumab in realtime fluorescent imaging and histologic processing of cutaneous squamous cell carcinoma (cSCC) in mice. Design. A near-infrared (NIR) fluorescent probe (IRDye800-CW) was covalently linked to a monoclonal antibodytargeting epidermal growth factor receptor (panitumumab) or nonspecific IgG and injected into mice bearing flank xenografts from a cSCC cell line (SCC-13 or SRB-12; n = 7), human splitthickness skin grafts (STSGs; n = 3), or a human tumor explant (n = 1). The tumor and lymph nodes were imaged and dissected using fluorescence guidance with the SPY imaging system and verified with a charge-coupled NIR system. An NIR scanning device (Odyssey) was used to measure fluorescence intensity in histological sections. Subjects. Immunodeficient mice. Setting. In vivo and in vitro imaging lab. Results. Tumor tissue could be delineated from the human STSG with tumor-to-background ratios of 4.5 (Pearl) and 3.4 (SPY). Tumor detection was substantially improved with panitumumab-IRDye800 compared with IgG-IRDye800. Biopsies positive for fluorescence were assessed by histology and immunohistochemistry (n = 18/18) to confirm the presence of tumor, yielding a 100% sensitivity. Biopsies of nonfluorescent tissue negative for malignancy (n = 18/18) yielded a specificity of 100%. Furthermore, the SPY system was able to detect residual disease as small as 200 mm in diameter. In addition, the Odyssey confirmed fluorescence of microscopic disease (in tumor samples of frozen and paraffin-embedded histologic specimens) but not in adjacent noncancerous tissue. Conclusions. These data suggest panitumumab-IRDye800 may have clinical utility in detection and removal of subclinical cSCC using Food and Drug Administrationapproved imaging hardware. © 2013 American Academy of Otolaryngology Head and Neck Surgery Foundation.