Background: Investigations for prolonged jaundice vary in complexity, especially in well neonates. The aim was to ascertain the causes of prolonged jaundice in well neonates and to analyse the extent and consistency of investigations performed. Methods: We performed a retrospective review of nurse-led prolonged jaundice screening in our children’s assessment unit from 2013 to 2016. The proforma included clinical assessment and recommended investigations. Data from electronic records and laboratory findings were compiled for analysis. Results: A total of 116 infants aged 14-73 days were referred for screening, with 100% utilization of the screening proforma. All patients had unconjugated hyperbilirubinaemia with normal urine and stool colour. Of the 113 with a full blood count performed, 74 (65.5%) were normal, with neutropenia in 32 (28.3%) and 1 blood film showing spherocytosis with haemolysis. Urine culture was performed in 106 infants, yielding 2 pure-growth cultures in infants subsequently treated for urinary tract infection (UTI) (1%). Seventy-four infants had one or more additional tests. The causes of prolonged jaundice identified were breast-milk jaundice (97), feeding difficulties (7), UTI (2) and hereditary spherocytosis (1). Conclusions: The most common cause of prolonged jaundice identified was breast-milk jaundice. The eldest referred infant was 73 days old, demonstrating a need for increased community awareness of guidelines to facilitate prompt referral. Additional investigations yielded little diagnostic value and we propose that in well neonates, following clinical evaluation, investigations may safely be reduced to full blood count and split bilirubin.