Abstract: BACKGROUND: Neonatal hypoglycemia management in the first 48 hours is guided by the American Academy of Pediatrics (AAP) and Pediatric Endocrine Society (PES) recommendations. Our aim was to determine the incidence of hypoglycemia via point of care test (POCT) on the 2nd day of life (DOL) among healthy, asymptomatic neonates regardless of risk factors. METHODS: In this prospective observational study, preprandial point of care glucose concentration was measured on the 2nd DOL in 150 healthy, asymptomatic neonates in the newborn nursery. We used 50 mg/dl (2.8 mmol/L) as the hypoglycemia threshold based on PES recommendations. RESULTS: The incidence of hypoglycemia on the second DOL was 10% among asymptomatic neonates (no risk factors = 8%; late preterm birth (LPT) + small for gestational age (SGA) = 16%; large for gestational age (LGA) + infant of diabetic mother (IDM) = 6%). SGA + LPT neonates accounted for the majority of the hypoglycemic cases (53.3%) and exhibited a trend towards the lowest glucose concentration (p = 0.09). CONCLUSION: The incidence of hypoglycemia on DOL 2 among asymptomatic neonates is high and of unclear significance in the absence of dedicated neurodevelopmental follow-up.