Polymyxin B (PMB) is the final option for treating multidrug-resistant Gram-negative bacterial infections. The acceptable pharmacokinetic/pharmacodynamic target is an area under the concentration–time curve across 24 h at a steady state (AUCss,24h) of 50–100 mg·h/L. The limited sampling strategy (LSS) is useful for predicting AUC values. However, establishing an LSS is a time-consuming process requiring a relatively dense sampling of patients. Further, given the variability among different centers, the predictability of LSSs is frequently questioned when it is extrapolated to other clinical centers. Currently, limited data are available on a reliable PMB LSS for estimating AUCss,24h. This study assessed and validated the practicability of LSSs established in the literature based on data from our center to provide reliable and ready-made PMB LSSs for laboratories performing therapeutic drug monitoring (TDM) of PMB. The influence of infusion and sampling time errors on predictability was also explored to obtain the optimal time points for routine PMB TDM. Using multiple regression analysis, PMB LSSs were generated from a model group of 20 patients. A validation group (10 patients) was used to validate the established LSSs. PMB LSSs from two published studies were validated using a dataset of 30 patients from our center. A population pharmacokinetic model was established to simulate the individual plasma concentration profiles for each infusion and sampling time error regimen. Pharmacokinetic data obtained from the 30 patients were fitted to a two-compartment model. Infusion and sampling time errors observed in real-world clinical practice could considerably affect the predictability of PMB LSSs. Moreover, we identified specific LSSs to be superior in predicting PMB AUCss,24h based on different infusion times. We also discovered that sampling time error should be controlled within −10 to 15 min to obtain better predictability. The present study provides validated PMB LSSs that can more accurately predict PMB AUCss,24h in routine clinical practice, facilitating PMB TDM in other laboratories and pharmacokinetics/pharmacodynamics-based clinical studies in the future.