Background: The distinction between tumor and healthy tissues is complicated in the areas previously subjected to radiation therapy (RT). This is related to the fact that tissues can undergo delayed and irreversible deterioration such as inflammation, vascular alteration and fibrosis. The trials related to the fluorescence –guided surgery (FSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) patients, previously subjected to RT, have not yet been reported. The present study addresses for the first time the possibilities of tumor near-infrared (NIR) imaging using Indocynaine Green (ICG) in irradiated areas. Methods: Four patients with histologically confirmed HNSCC were included in this study. All included patients were previously treated with RT with at least 50 Gy. RT-radiation fields from original treatment fully encompassed the second tumor or recurrence. ICG was injected via cephalic vein 45 min before the images were captured using a NIR camera system Artemis. The images were also captured before ICG injection serving as background signal. The fluorescence intensity measurements were carried out using specially designed software. Results: ICG fluorescence clearly demonstrated a significant difference in fluorescence intensity between healthy and tumor tissues in 2 of 4 patients. Histology post-resection analysis confirmed a complete tumor resection with safe surgical margins. No difference between tumor and surrounding healthy tissue was detected in patients with an epidermoid carcinoma developed from sclerohypertrophic lichen. Conclusions: In our pilot study, we clearly established the feasibility of using NIR FGS with ICG to delineate tumor and healthy tissues in irradiated areas in infiltrating lichen-free tumors.