Background: Ultrafiltration (UF) is a conventional method for isolating the protein-unbound plasma fractions of therapeutic drugs. However, the ideal UF conditions for specific compounds remain largely unexplored. By comparing UF-derived unbound concentrations with the corresponding results obtained using a reference method, the authors sought to identify appropriate UF conditions for cefotaxime, cloxacillin, flucloxacillin, and piperacillin. Methods:In vitro microdialysis (MD) with a no-net-flux approach was used as a reference method for plasma protein separation, for which UF performance was assessed. Four levels of relative centrifugal force (2500-11,290g) and 2 levels of temperature (37 vs. 228C) during 10 minutes of UF centrifugation were evaluated. Ultrafiltrates and reference microdialysates were analyzed using liquid chromatography-tandem mass spectrometry to obtain unbound concentrations. After identifying the appropriate UF conditions in the spiked plasma samples, exploratory analyses of clinical samples (n = 10 per analyte) were performed.Results: Of the evaluated UF alternatives, the best overall agreement with the MD-derived reference concentrations was obtained with 11,290 g UF performed at 228C. For cloxacillin specifically, 378C UF yielded better agreement than 228C UF at 11,290 g. Clinical sample analyses indicated minimal differences between 228C and 378C at 11,290 g UF for cefotaxime and piperacillin. However, consistently lower levels of unbound cloxacillin (median: 223%, IQR: 219% to 224%) and flucloxacillin (median: 227%, IQR: 221 to 234%) were observed after UF at 228C versus 378C. Conclusions:For the evaluated UF device, 10 minutes of 11,290 g UF at 228C is appropriate for flucloxacillin, cefotaxime, and piper-acillin, and can arguably be justified for cloxacillin as well for laboratory practice purposes. Maintenance of 378C during highcentrifugal UF may lead to overestimation, particularly for unbound flucloxacillin.