Insulinomas are rare endocrine tumors that are usually diagnosed by inappropriate elevations in insulin and C-peptide during hypoglycemia. We report a case of a surgically confirmed insulinoma diagnosed by a mild elevation in proinsulin with suppressed insulin and C-peptide at the time of hypoglycemia during a supervised fast. A supervised fast with serial measurements of plasma glucose, insulin, and C-peptide was performed in a patient with documented hypoglycemia. Proinsulin was measured at the beginning and end of the fast. Tumor localization was accomplished with spiral CT, magnetic resonance imaging, and endoscopic ultrasound. Minimally invasive tumor resection was performed. The presence of an insulinoma was confirmed on the basis of a minimally elevated proinsulin level with a suppressed insulin level at the time of symptomatic hypoglycemia. Tumor resection was performed without complications, resulting in resolution of the hypoglycemia. This case demonstrates the importance of measuring proinsulin as a routine component of the 72-hour fast for detection of an insulinoma. Even mild elevations in circulating proinsulin can be an independent indicator of aberrant insulin secretion during hypoglycemia. Once the diagnosis of insulinoma is made and tumor localization is achieved, minimally invasive tumor resection is a safe and effective treatment modality.