Abstract Purpose Intra-operative management of the surgical margin in patients diagnosed with head and neck squamous cell carcinoma (HNSCC) remains challenging as surgeons still have to rely on visual and tactile information. Fluorescence-guided surgery using tumor-specific imaging agents can assist in clinical decision-making. However, a standardized imaging methodology is lacking. In this study, we determined whether a standardized, specimen-driven, fluorescence imaging framework using ONM-100 could assist in clinical decision-making during surgery. Procedures Thirteen patients with histologically proven HNSCC were included in this clinical study and received ONM-100 24 ± 8 h before surgery. Fluorescence images of the excised surgical specimen and of the surgical cavity were analyzed. A fluorescent lesion with a tumor-to-background ratio (TBR) > 1.5 was considered fluorescence-positive and correlated to standard of care (SOC) histopathology. Results All six tumor-positive surgical margins were detected immediately after excision using fluorescence-guided intra-operative imaging. Postoperative analysis showed a median TBR (±IQR) of the fluorescent lesions on the resection margin of 3.36 ± 1.62. Three fluorescence-positive lesions in the surgical cavity were biopsied and showed occult carcinoma and severe dysplasia, and a false-positive fluorescence lesion. Conclusion Our specimen-driven fluorescence framework using a novel, pH-activatable, fluorescent imaging agent could assist in reliable and real-time adequate clinical decision-making showing that a fluorescent lesion on the surgical specimen with a TBR of 1.5 is correlated to a tumor-positive resection margin. The binary mechanism of ONM-100 allows for a sharp tumor delineation in all patients, giving the surgeon a clinical tool for real-time margin assessment, with a high sensitivity.