The study was aimed to evaluate the performance of a newly developed non-invasive and noncontact bilirubin measurement device (AJo-neo) as an alternative to the conventional invasive biochemical method of total serum bilirubin (TSB) estimation in preterm and term neonates suffering from hyperbilirubinemia associated with risk factors, and/or undergoing phototherapy. The safety and efficacy of the device were assessed in 1968 neonates with gestational ages ranging from 28 to 41 weeks and suffering from incidences of hyperbilirubinemia. Linear regression analysis showed a good correlation between AJO-Neo and the conventional method of TSB (Pearson's coefficient, r = 0.79). The small bias (0.27 mg/dL) and limits of agreements (− 3.44 to 3.99 mg/dL) were within the range of clinical acceptance. The device was also precise in the measurement of bilirubin levels in all subgroups of the study. The receiver operator curve (ROC), that takes account of both sensitivity and specificity of a device showed high efficacy of the device (area under the curve, AUC = 0.83) in the detection of bilirubin. While monitoring the bilirubin level during phototherapy, the device indicated promising results showing good agreement with TSB. Specificities and sensitivities of the device indicated a much higher accuracy in neonates with associated risk factors for hyperbilirubinemia. Hence, the newly developed device (AJO-Neo) is reliable in measuring bilirubin level in preterm, and term neonates irrespective of gestational or postnatal age, sex, risk factors, feeding behavior or skin color. Neonatal jaundice or Icterus neonatorum affects more than 60% of the term and 80% of preterm newborns in the first week of life 1-3 due to excessive production of bilirubin, and the inability of the newly developed liver to excrete it 4. Although benign at low concentrations, persistently elevated levels of bilirubin can cause severe neurotoxicity termed as kernicterus, which may lead to significant morbidity and mortality 5,6. Thus, proper monitoring of the bilirubin level in newborns is mandatory as per the guideline of American Academy of Pediatrics (AAP) to ensure appropriate management 7. The contemporary method of detection of serum bilirubin