Background and ObjectivesMolecularly targeted fluorescent molecules may help detect tumors that are unseen by traditional white‐light surgical techniques. We sought to evaluate a fluorescent ratiometric activatable cell penetrating peptide (RACPP) for tumor detection in a transgenic model of PTC.MethodsThirteen BRAFV600E mice with PTC were studied—seven injected intravenously with RACPP, four controls with saline. Total thyroidectomy was performed with microscopic white‐light visualization. Fluorescent imaging of post‐thyroidectomy fields was performed, and tissue with increased signal was removed and evaluated for PTC. Final samples were analyzed by a pathologist blinded to conditions. Vocal cord function was evaluated postoperatively with video laryngoscopy.ResultsThe average in situ ratiometric (Cy5/Cy7) thyroid tumor‐to‐background contrast ratio was 2.27 +/− 0.91. Fluorescence‐guided clean‐up following thyroidectomy identified additional tumor in 2 of 7 RACPP animals (smallest dimension 1.2 mm), and decreased the number of animals with residual tumor from 4 to 3. All retained tumor foci on final pathology were smaller than 0.76 mm. Intact vocal abduction was present in all of the RACPP animals.ConclusionsRACPPs successfully targeted PTC in a transgenic thyroidectomy model, and allowed for residual tumor detection that reduced positive margins beyond what was possible with white‐light surgery alone. J. Surg. Oncol. 2016;113:138–143. © 2016 Wiley Periodicals, Inc.