Background: Infants born to diabetic mothers face an increased risk of hypoglycemia, necessitating reliable predictive markers for early intervention. This study aimed to measure C-Peptide in umbilical cord blood, to determine whether it can be used as a predictor of hypoglycemia in infants of diabetic mothers. Methods: A total of 50 infants born to diabetic mothers were studied, with 25 experiencing hypoglycemia and 25 remaining normoglycemic. Detailed histories, comprehensive examinations, and multi-level investigations were conducted, including C-peptide measurement from umbilical cord blood and monitoring of glucose levels postnatally. Results: In the hypoglycemic group, mean cord C-peptide levels were significantly higher (5.2 ± 0.55 ng/ml) compared to the normoglycemic group (1.71 ± 0.62 ng/ml). A significant negative correlation was found between cord C-peptide levels and the incidence of hypoglycemia. Additionally, a positive correlation existed between cord C-peptide levels and birth weight. The sensitivity of cord C-peptide in predicting hypoglycemia was 90%, with a specificity of 88% and a cut-off value of 4.2. Conclusion: Cord C-peptide levels in infants of diabetic mothers show promise as a predictive biomarker for hypoglycemia, demonstrating high sensitivity and specificity.