Physiological jaundice is the most common condition that requires medical attention and hospital readmission of neonates. About 60% of term and 80% of preterm babies develop jaundice in the first week of life. Furthermore, about 10% of breastfed babies stay with jaundice until the age of 1 month. In the current study, a total of 139 neonates were evaluated for physiological jaundice in which 89 (64%) were male neonates and 50 (36%) were female neonates. Blood samples were collected under aseptic conditions. All samples were performed on Roche/Hitachi COBASc, based on the spectrophotometric technique. The mean SD (15.867±5.57) was calculated for the total, direct, and indirect bilirubin in neonates who were divided into 3 groups based on the number of days, that is, 1-2, 3-5, and 6-22 days, respectively. For indirect bilirubin in the age group of 1-2, ±SD was 13.80±5.41 in males, whereas it was ±10.40 in females, while the p-value < 0.01 was significant. Hyperbilirubinemia is associated with bilirubin due to a higher mass of red blood cells. It was found that the total, direct, and indirect bilirubin significantly increased in the 1st to 5th day of life after birth with male neonates being more vulnerable to pathological jaundice as compared to female neonates.