Objective: Establish the feasibility of optimizing PEEP (PEEPOPT) using ventilator software in infants receiving prolonged invasive mechanical ventilation. Methods: A retrospective review of infants who underwent PEEPOPT studies. Paired-t test was used to compare dynamic lung compliance (Cdyn), tidal volume (VT), and oxygen saturation (SpO2) before and after PEEPOPT studies. Results: Fifteen infants with bronchopulmonary dysplasia and/or receiving prolonged invasive mechanical ventilation were included with a median gestational age of 24 4⁄7 weeks and postmenstrual age of 40 1⁄7 weeks at the time of the PEEPOPT study. PEEPOPT was different from clinically set PEEP (PEEPSET) in 14⁄15 infants; PEEPOPT was higher in 27% and lower in 67% of infants (–2 to +5cmH2O). Cdyn improved for most infants by 0.23 (0.01–0.60) mL/H2O (p < 0.001). VT increased for most infants by 1.5 (–0.5 to 3.4) mL/kg (p < 0.001), and SpO2 increased by 2% (–1 to 12%). Conclusion: Assessment of PEEPOPT using our standardized protocol is feasible, non-invasive, and provides a low-cost bedside tool. PEEPOPT differed from PEEPSET in most subjects, highlighting the importance of evaluating PEEPOPT.