In the United States, approximately 13% of the annual birth cohort is born preterm (<37 weeks gestational age). These infants are at high risk of systemic infections, and most are treated with antimicrobial agents. In spite of their common use, antimicrobials lack pharmacokinetic (PK) data in preterm infants. Extrapolation of dosing information from older children and adults to preterm infants often results in therapeutic failures or unwanted toxicities. Therefore, PK studies in this population are needed. Unique challenges posed by preterm infants, however, limit the ability to evaluate drug disposition in this population. Novel, minimal-risk methods can provide the platform to overcome these challenges and optimize antimicrobial dosing. In this proposal, several of these methods were employed to deliver antimicrobial dosing recommendations specifically designed for preterm infants. Two commonly used antimicrobials, piperacillin and metronidazole, were used to evaluate the utility of these methods. A multiplex liquid chromatography-tandem mass spectrometry assay for the simultaneous quantification of ampicillin, piperacillin, tazobactam, meropenem, acyclovir, and metronidazole in ultra-low (<50 uL) human plasma was developed and validated. This method was used to analyze drug concentration data from sparse, scavenged, and dried blood spot PK samples collected from preterm infants <32 weeks gestational age at birth. The scavenged concentration data were analyzed by population PK methodologies and used to derive PK parameters and identify covariates (serum creatinine and postmenstrual age) that explain inter-individual variability in piperacillin and metronidazole clearance. When compared to historical data from small studies using traditional PK sampling, piperacillin concentrations were 2-10-fold lower in the scavenged sampling approach, which prevented the determination of optimal dosing recommendations for piperacillin. In contrast, scavenged sampling proved successful for metronidazole and allowed for the development of a simplified, postmenstrual age-based dosing regimen. Piperacillin and tazobactam dried blood spot sampling proved successful as a proof-of-concept application. Dried blood spot piperacillin and tazobactam concentrations can be used as surrogate for plasma measurements. Novel, minimal-risk methods are powerful tools to evaluate the pharmacokinetics/pharmacodynamics of antimicrobials in preterm infants. The success of these methods is drug-dependent, and they should be systematically studied prior to universal implementation.