G6PD deficiency is the most common enzymatic aberration, affecting more than a million people worldwide. If severe cases at birth are not diagnosed and treated promptly, they can develop into cerebral jaundice and hyperbilirubinemia. Hyperbilirubinemia is caused by an imbalance between increased bilirubin production and ineffective excretion of bilirubin. This can result in the neurotoxicity of bilirubin, kernicterus, and fatal mental retardation. Besides, It has been speculated that the presence of a high number of reticulocytes in newborns interferes with the diagnosis of G6PD deficiency since reticulocytes contain higher amounts of G6PD enzyme than mature erythrocytes. Therefore, the purposes of this study were to assess the effect of reticulocytosis in the determination of blood G6PD activity in Thai newborns by using an automated UV enzymatic assay, to examine the association between the levels of G6PD activity and bilirubin, and to validate the performance of this assay for the detection of G6PD deficiency in newborn blood samples. Our data revealed that the prevalence of G6PD deficiency was 10.0% in Thai male newborns and 2.3% in females. The minor allele frequency (MAF) of common mutation in the study population; G6PD ViangchanG871A was 0.066. Compared with normal newborns after controlling for thalassemia and hemoglobinopathies, the reticulocyte counts in newborns with G6PD deficiency were significantly higher than that of normal newborns (p<0.001). There was no correlation between G6PD activity and reticulocyte counts in subjects with G6PD deficiency (r=-0.019, p=0.881), whereas the levels of G6PD activity in normal newborns were positively correlated with the percentage of reticulocytes (r=0.327, p<0.001). Moreover, our results showed that the total serum bilirubin and indirect bilirubin in all newborns with intermediate and deficient G6PD were significantly higher than that of normal G6PD newborns. The level of agreement in the detection of G6PD deficiency was 0.999, while the area under the curve of receiver operating characteristic (AUC) demonstrated that the automated UV enzymatic assay had 98.4% sensitivity, 99.5% specificity, 92.4% positive prediction value (PPV), 99.9% negative predictive value (NPV), and 99.4% accuracy. We report that reticulocytosis, commonly observed in newborns, does not have a statistically significant effect on the diagnosis of G6PD deficiency in newborns by both qualitative and quantitative methods. G6PD-deficient newborns had high levels of indirect bilirubin, which might cause serious consequences for newborns.� �