Objective: To determine the frequency of glucose-6-phosphate dehydrogenase (G6PD) deficiency in neonates presenting with indirect hyperbilirubinemia. Methods: A prospective cross-sectional study was conducted at Neonatal intensive care unit (NICU) of National Institute of Child Health, Karachi from August 2020 to July 2021. All neonates with either gender born with indirect hyperbilirubinemia >5mg/dl admitted in NICU were consecutively enrolled. G6PD deficiency along with baseline and laboratory characteristics were observed. The presence of G6PD level of <1.3 U/g Hb was labeled as G6PD deficiency. Results: Of 228 neonates, the mean age was 8.58 ±3.68 days. There were 153 (67.1%) males and 75 (32.9%) females. The frequency of G6PD deficiency was found to be 72 (31.6%). The mean weight was significantly higher among neonates with G6PD deficiency as compared to the patients with no G6PD deficiency, i.e., 2.53 ±0.48 vs. 2.37 ±0.56 (p-value 0.035). A significant difference of hemoglobin level (p-value <0.001), serum total bilirubin level (pvalue <0.001), and serum indirect bilirubin level (p-value0.001) was observed in between G6PD deficient and nondeficient neonates. Increased reticulocyte count was also found significantly higher in neonates with G6PD deficiency as compared to the neonates without G6PD deficiency, i.e., 69 (95.8%) and 34 (21.8%) respectively (pvalue <0.001). Conclusion: G6PD deficiency was considerably prevalent in neonates with indirect hyperbilirubinemia. Moreover, hemoglobin level, serum total bilirubin level, serum indirect bilirubin level, and increased reticulocyte count was also observed significant contributing factors for G6PD deficiency.