Piperacillin is commonly used off‐label in neonates for the treatment of bacterial infections. This study aimed to assess a dried blood spots (DBS)‐based microsampling strategy for supporting population pharmacokinetics and treatment optimization of piperacillin in Chinese neonates. DBS samples from neonatal patients were collected at predefined intervals. Drug blood concentrations were quantified using a validated ultra‐high‐performance liquid chromatography‐tandem mass spectrometry method. A population pharmacokinetic model was developed using a nonlinear mixed‐effects modeling approach. The pharmacokinetic/pharmacodynamics (PK/PD) target was 75% of the time with the unbound drug plasma concentration above the minimum inhibitory concentration (fT>MIC), with a toxicity threshold of unbound drug plasma trough concentration above 64 mg/L. A total of 45 piperacillin samples from 24 neonates were collected. The pharmacokinetics of piperacillin was described using a one‐compartment model with postmenstrual age (PMA) as the most significant covariate on clearance. Simulations showed that dosing regimens achieving >90% PK/PD target attainment with <10% risk of possible toxicity were: PMA 33‐35 weeks (50 mg/kg q12h), 35‐37 weeks (50 mg/kg q8h), and 37‐41 weeks (50 mg/kg q6h). In conclusion, Using DBS sampling, we developed a population pharmacokinetic model of piperacillin in Chinese neonates, incorporating PMA to determine optimal dosing regimens.