In neonates, propofol mainly undergoes hydroxylation to quinol metabolites with only limited glucuronidation. The aim of this study is to search for covariates of neonatal propofol biotransformation based on 24 h urine collections. In neonates receiving an intravenous propofol bolus for short procedural sedation, urine was collected during 24 h. Urinary propofol metabolites [propofol glucuronide (PG), 1- and 4-quinol glucuronide (QG)] were determined using high-performance liquid chromatography after a dual-step solid phase extraction combined with ultraviolet and fluorescence detection. Propofol metabolites, their contribution to total metabolite elimination and propofol glucuronide/quinol glucuronide (PG/QG) ratio were determined. The impact of continuous [postmenstrual age (PMA), postnatal age (PNA), body weight, propofol dose, creatinaemia] and dichotomous variables [PNA ≤ 7 days (yes/no), PNA ≥ 10 days (yes/no), hyperbilirubinaemia (yes/no), cardiopathy (yes/no)] on PG/QG ratio and on patients with low (≤10 %) vs. high (>10 %) urinary PG recovery were examined. Thirty-two neonates were included. Median total propofol metabolite recovery was 40.95 (2.01-129.81) % with PG/QG ratio 0.44 (0.01-5.93). PNA (dichotomous 7 days as well as 10 days) was a significant covariate of PG/QG ratio. Late PNA more frequently resulted in high urinary PG fraction. Significance was more pronounced with PNA 10 days as cut-off point for early neonatal life compared to 7 days. Age 10 days is pivotal in early life propofol metabolism. This confirms earlier documented propofol clearance studies. This is the first report of the modified quantification assay used to determine urinary propofol metabolites in neonates.