To date, many population pharmacokinetic models of antibiotics have been developed using blood sampling data after the fourth or fifth dose, which represents steady-state levels. However, if a model developed using blood sampled after the first dose is equivalent to that using blood sampled after the fourth dose, it would be advantageous to utilize the former. The aim of this study was to investigate the effect of blood sampling after the first and/or fourth drug administration on the accuracy and precision of parameter estimates. A previously reported robust, two-compartment model of vancomycin was used for simulation to evaluate the performances of the parameter estimates. The parameter estimation performances were assessed using relative bias and relative root mean square error. Performance was investigated in 72 scenarios consisting of a combination of two blood sampling periods (the first and fourth dose), two total clearances, three infusion times, and four sample sizes. The population pharmacokinetic models from data collected at the first dose and those collected at the fourth dose produced parameter estimates that were similar in accuracy and precision. This study will contribute to increasing the efficiency and simplicity of antibiotic pharmacokinetic studies.