In human plasma, the total concentration of non-protein binding (NPB) drugs is equal to the free drug concentration because NPB drugs do not or hardly bind to plasma proteins. Thus, centrifuge ultrafiltration (CF-UF) has been used in the determination of the concentration of NPB drugs in human plasma. However, with only a common centrifugation, the recovery and the reproducibility were not as excellent as expected. In addition, we discovered that the values of the volume ratio of ultrafiltrate to sample solution (Vu/Vs) were different and could not be well controlled, which may affect the determination of the drug concentration. The problem also affected the determination of other NBP drugs. In the present work, we used biapenem as a representative drug to study the effect of Vu/Vs on the analysis of NPB drugs concentration in human plasma. The results showed that a Vu/Vs value of less than 0.4 had no effect on the analysis of free drug concentration, while a Vu/Vs value of more than 0.4 was associated with increased recovery rate and overestimation of drug concentration. Therefore, to maintain a Vu/Vs value of less than 0.4 and even at a constant value is the key to accurately determine the concentration of NPB drugs in plasma. Fortunately, with an HFCF-UF device, the Vu/Vs could be well controlled and kept at 0.08 in this study. The recovery rates were almost 100% and the analysis precision was greatly improved. In pharmacokinetics studies, this method was successfully employed to determine the concentration of biapenem with excellent accuracy and reproducibility. HFCF-UF may become a feasible platform for the determination of NPB drugs.