Pharmacokinetic models rarely undergo external validation in vulnerable populations such as critically ill infants, thereby limiting the accuracy, efficacy, and safety of model-informed dosing in real-world settings. Here, we describe an opportunistic approach using dried blood spots (DBS) to evaluate a population pharmacokinetic model of metronidazole in critically ill preterm infants of gestational age (GA) ≤31 weeks from the Metronidazole Pharmacokinetics in Premature Infants (PTN_METRO, NCT01222585) study. First, we used linear correlation to compare 42 paired DBS and plasma metronidazole concentrations from 21 preterm infants mean (SD): post natal age 28.0 (21.7) days, GA 26.3 (2.4) weeks. Using the resulting predictive equation, we estimated plasma metronidazole concentrations (ePlasma) from 399 DBS collected from 122 preterm and term infants mean (SD): post natal age 16.7 (15.8) days, GA 31.4 (5.1) weeks from the Antibiotic Safety in Infants with Complicated Intra-Abdominal Infections (SCAMP, NCT01994993) trial. When evaluating the PTN_METRO model using ePlasma from the SCAMP trial, we found that the model generally predicted ePlasma well in preterm infants with GA ≤31 weeks. When including ePlasma from term and preterm infants with GA >31 weeks, the model was optimized using a sigmoidal Emax maturation function of postmenstrual age on clearance and estimated the exponent of weight on volume of distribution. The optimized model supports existing dosing guidelines and adds new data to support a 6-hour dosing interval for infants with postmenstrual age >40 weeks. Using an opportunistic DBS to externally validate and optimize a metronidazole population pharmacokinetic model was feasible and useful in this vulnerable population.