Amprenavir is an HIV-1 protease inhibitor with high protein binding (90%) in human plasma. This study was designed to develop an assay to measure the concentration of unbound amprenavir, to study variation with time in patients, and to investigate whether ritonavir and lopinavir, other protease inhibitors that could be combined, interact with amprenavir protein binding in vitro. A reverse-phase high-performance liquid chromatography assay to UV detection was developed and validated to measure total amprenavir in plasma, and this method was adapted to quantitate low concentrations of unbound amprenavir in ultrafiltrate aqueous fluid. Equilibrium dialysis and ultrafiltration were used and compared with separate unbound fraction. The latter method was easier to use and was, therefore, subsequently adopted. In 10 patients who received amprenavir 600 mg bid combined with ritonavir, mean amprenavir free-fraction in plasma was 8.6% (range, 4.4-20%). When added to pooled human plasmas at concentrations close to those found in treated patients, the unbound amprenavir fraction was increased in the presence of lopinavir, but remained unaffected by ritonavir. It remains to be seen whether measurement of unbound concentrations, rather than total concentrations, could improve therapeutic drug monitoring.