Background: Neonatal hypoxic-ischemic encephalopathy (HIE) is a devastating condition resulting from a sustained lack of oxygen during birth. The aim of the present study was to evaluate serum concentrations of neuron-specific enolase (NSE) and the highly soluble glial protein S-100 as a marker of the severity of HIE. Methods: We conducted a case-control study for 38 newborns diagnosed with moderate to severe hypoxic-ischemic encephalopathy and compared with 80 healthy controls. Two blood samples were taken to measure NSE and S100 levels, one at the birth time and the other in 24 h after birth for both groups. Comparison between cases and controls was performed using Fisher's exact or Chi-square test and Mann-Whitney U test. P value < 0.05 was considered statistically significant. Results: In this study, the 118 neonates were studied in two groups, 38 neonates had asphyxia (case group) and 80 neonates were selected as the control group. The difference in S100 and NSE1 concentration was statistically significant between HIE cases and controls (P < 0.05) for the first taken sample. Conclusion: levels of brain-specific proteins, such as S100, and NES can be assessed to identify infants at the highest risk of brain damage.