To assess the impact of experimental conditions on free serum concentrations as determined by ultrafiltration and HPLC-DAD analysis in a wide range of antibiotics. Relative centrifugation force (RCF), temperature, pH and buffer were varied and the results compared with the standard protocol (phosphate buffer pH 7.4, 37°C, 1000 × g). Generally, at 10,000 × g the unbound fraction () decreased with increasing molecular weight, and was lower at 22°C. In unbuffered serum, the of flucloxacillin or valproic acid was increased, that of basic or amphoteric drugs considerably decreased. Comparable results were obtained using phosphate or HEPES buffer except for drugs which form metal chelate complexes. Maintaining a physiological pH is more important than strictly maintaining body temperature.