Published data on the unbound fraction of vancomycin in patient samples exhibit high variability. In the present study, a robust ultrafiltration method was developed and applied to 102 clinical samples from 22 intensive care unit patients who were treated with continuous infusion of vancomycin. A validated HPLC method was used for determination of total and unbound concentrations. The mean unbound fraction was 67.2% (standard deviation 7.5%, range 47.2–92.1%) and independent of total concentration of vancomycin or of albumin. The unbound fraction was significantly correlated (r = +0.67, P = .0009) with the renally filtered fraction (drug clearance/creatinine clearance), providing functional evidence for the validity of the measurements. Ultrafiltration proved to be susceptible to variations in the experimental conditions such as pH, temperature and centrifugal force. The measured unbound fraction increased from 60% at pH 6 to 100% at pH 9, from 57% at 4°C to 80% at 37°C, and was 76% at 1,000 g compared with 45% at 10,000 g. Lack of standardization may therefore partly explain the variable results reported in the literature.