<h4>Objectives</h4>The aim of this study was to evaluate the use of cathepsin-activated intraoperative near-infrared (NIR) imaging to detect insulinomas in dogs, a spontaneous large animal model for human disease.<h4>Materials and methods</h4>A prospective, pilot clinical trial was performed on dogs with naturally occurring insulinomas undergoing exploratory laparotomy. The gene discussed is CTSS; the disease is pancreatic insulinoma.