Neonatal jaundice is a common and severe disease in premature infants with Glucose-6-Phosphate Dehydrogenase (G-6-PD) deficiency. The World Health Organization (WHO) has recommended screening for G-6-PD deficiency in newborns for early recognition as well as to prevent unwanted outcomes in a timely manner. The present study aimed to assess a point-of-care, careSTARTTM G6PD biosensor as a quantitative method for the diagnosis of G-6-PD deficiency. Factors influencing the evaluation of G-6-PD enzyme activity were examined in 40 adults, including ethylenediaminetetraacetic acid (EDTA) anticoagulant, hematocrit concentration, storage temperature and time. Analytic performance of the careSTARTTM G6PD biosensor was evaluated in 216 newborns and compared with fluorescent spot test (FST) and standard quantitative G-6-PD enzyme activity (SGT) assay. The results of factors affecting the G-6-PD enzyme activity showed that the activity determined from finger-prick was not statistically different from venous blood (p = 0.152). The G-6-PD value was highly dependent on the hematocrit and rose with increasing hematocrit concentration. Its activity was stable at 4°C for 3 days. Reliability analysis between the careSTARTTM G6PD biosensor and SGT assay showed a strong correlation with a Pearson’s correlation coefficient of 0.82 and perfect agreement by intraclass correlation coefficient (ICC) of 0.90. Analysis of the area under the Receiver Operating Curve (AUC) illustrated that the careSTARTTM G6PD biosensor had 100% sensitivity, 96% specificity, 73% positive predictive value (PPV), 100% negative predictive value (NPV) and 97% accuracy at 30% of residual activity. While the diagnostic ability for identifying G-6-PD deficiency had 78% sensitivity, 89% specificity, 56% positive predictive value (PPV), 96% negative predictive value (NPV) and 88% accuracy when stratified by gender. The careSTARTTM G6PD biosensor is an attractive option as a point-of-care quantitative method for G-6-PD activity detection. Quantification of G-6-PD enzyme activity in newborns is the most effective approach for the management of G-6-PD deficiency to prevent severe jaundice and acute hemolysis.