Total plasma concentrations are used for therapeutic drug monitoring of antiretroviral drugs, whereas antiviral activity is expected to depend on unbound concentrations. The determination of free (unbound) concentrations by ultrafiltration may be flawed by the irreversible adsorption of many drugs onto the membrane filters and plastic components of the device. The authors describe a modified ultrafiltration method enabling the accurate measurement of unbound concentrations of 10 antiretroviral drugs by liquid chromatography-tandem mass spectroscopy, which circumvents the problem of loss by adsorption in the early ultrafiltration fractions. The method was applied to assess the variability of free fractions of antiretroviral drugs during routine therapeutic drug monitoring in 144 patients with HIV. In in vitro experiments, ultrafiltrate collected in four fractions (0-8, 8-16, 16-24, and 24-30 minutes) gave much lower and more variable free drug concentrations in the first ultrafiltrate fraction than in the last three fractions for lopinavir, nelfinavir, saquinavir, tipranavir, and efavirenz. In the last two fractions, free concentrations remained constant, indicating saturable adsorption. The adsorption was modest for indinavir, amprenavir, and ritonavir, and unnoticeable for atazanavir and nevirapine. Free fraction values obtained with this modified ultrafiltration method reveal substantial interindividual variability, suggesting that monitoring unbound antiretroviral drug concentrations may increase its clinical usefulness, especially for lopinavir, saquinavir, and efavirenz.