Background <p>Drug-drug interactions (DDIs) are a critical concern in neonatal intensive care units (NICUs) due to polypharmacy and the physiological vulnerability of neonates. This study aims to evaluate potential DDIs (pDDIs) using two different decision support software (DSS), Lexidrug™ and Micromedex^®.</p> Methods <p>This retrospective study included neonates admitted to a tertiary NICU between January 1, 2022, and January 1, 2023, who received at least two systemically administered medications and stayed for ≥ 24&#xa0;h. Patients with missing prescriptions or readmissions were excluded. pDDIs were assessed using two different DSS: Lexidrug™ and Micromedex^®. pDDIs were categorized by severity, and D/X (major/contraindicated) or equivalent levels were considered clinically significant. A neonatologist and clinical pharmacist independently evaluated all identified pDDIs for clinical relevance. The consistency between DSS outputs was analyzed using Kendall’s W and Cohen’s kappa statistics. Chi-square and Mann–Whitney U tests were used to evaluate risk factors. A network map of drug interactions was also created using Gephi software.</p> Results <p>The study included 280 patients. The gestational age median (interquartile range [IQR]) was 36 (34–38) weeks, and birth weight median (IQR) was 2745 (2181.2–3250) g. According to Lexidrug™ and Micromedex^® databases, any level pDDIs were found in 151 (53.9%) and 127 (45.4%) patients, respectively. No patient had X/contraindicated level pDDIs. D/major level pDDIs were found in 95 (33.9%) and 71 (25.4%) patients, respectively. Vancomycin, gentamicin, and fentanyl were the most commonly involved agents in pDDIs. There was a very weak concordance between the two DSS for pDDIs at the X/contraindicated and D/major levels and a weak concordance for total pDDIs (Kendall’s W values; 0.004; 0.005; 0.249, respectively). According to the consensus, 7 (22.5%) and 9 (14.7%) of the different pDDI pairs were clinically significant according to Lexidrug™ and Micromedex^®, respectively.</p> Conclusion <p>pDDIs were detected in a significant proportion of patients in the NICU. This study revealed weak agreement between different DSS used to assess pDDIs in the NICU. The findings underscore the importance of collaboration between clinical pharmacists and neonatologists when interpreting DSS results. Further prospective, multicenter cohort studies are warranted to validate these findings and improve DDI management strategies in neonatal populations.</p> Trial registration <p>Not applicable.</p>