Objective: Methods for measuring protein binding of drugs generally require direct measurement of the concentration of unbound drug and thus may require a highly sensitive assay. In vivo ultrafiltration has been used to determine protein binding of endogenous substances. We have examined its value for measuring protein binding of drugs because it requires measurement of only the concentration of total drug, not unbound drug, in plasma. Methods: The protein binding of aspirin and its metabolite salicylate was measured in 29 healthy subjects 20 minutes after a single oral dose of 600 mg soluble aspirin, by the new method, in vivo ultrafiltration, as well as by a standard method, in vitro ultracentrifugation. Results: The data for salicylate were examined systematically to determine the optimal method of determining estimates of protein binding by in vivo ultrafiltration. Estimates of protein binding of salicylate were 81.7% ± 10.1% (mean ± SD) by the in vivo method and 81.6% ± 11.3% by in vitro ultracentrifugation. Bland-Altman analysis of agreement showed that within-individual differences in percentage of protein binding determined by the two methods did not differ significantly from zero (mean difference, 0.07%; 95% confidence interval, -2.33 to +2.46). There was a highly significant correlation between estimates of protein binding by the two methods (r = 0.82; P = 0.001). Protein binding of aspirin was estimated at 58.3% ± 9.6% by in vivo ultrafiltration and could not be estimated by in vitro ultracentrifugation because the concentration of unbound aspirin in plasma was below the limit of detection for the assay. Conclusion: In vivo ultrafiltration can be used to measure protein binding of drugs and has potential advantages over conventional methods. A sensitive assay may not be required because the unbound drug need not be measured, measurement in vivo may maintain more physiologic conditions, and it may be useful in measuring protein binding of drugs that are degraded rapidly in vitro.