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To evaluate neonatal abstinence syndrome (NAS) treatment outcomes achieved using an optimized methadone weaning protocol developed using pharmacokinetic (PK) modeling compared with standard methadone weaning.This pre-post cohort study evaluated 360 infants who completed pharmacologic treatment for NAS with methadone as inpatients at 1 of 6 nurseries in southwest Ohio between January 2012 and March 2015. Infants were initially treated with a standard methadone weaning protocol (n = 267). Beginning in July 2014, infants were treated with a revised methadone weaning protocol developed using PK modeling (n = 93). Linear mixed models were used to calculate adjusted mean primary outcomes, including total duration of methadone treatment, total administered methadone dosage, and length of inpatient hospital stay, which were compared between weaning protocols. The use of adjunctive therapy for NAS treatment was examined as a secondary outcome.Infants who received NAS treatment with the revised protocol experienced a shorter duration of methadone treatment (13.1 vs 16.4 days; P < .001) and shorter duration of inpatient treatment (18.3 vs 21.7 days; P < .001) compared with infants receiving standard methadone weaning. No difference was observed in total methadone dosage administered (0.52 vs 0.52 mg/kg; P = .97) or in the use of adjunctive therapy (22.6% vs 25.5%; P = .68) between groups.Refinement of a standard methadone weaning protocol using PK modeling was associated with reduced duration of opioid weaning and shortened length of stay for pharmacologic treatment of NAS.