The use of microdialysis sampling to study the binding of drugs to plasma proteins was evaluated. Microdialysis sampling is accomplished by placing a short length of dialysis fiber in the sample and perfusing the fiber with a vehicle. Small molecules in the sample, such as drugs, diffuse into the fiber and are transported to collection vials for analysis. Larger molecules, such as proteins and protein-bound drugs are excluded by the dialysis membrane. Microdialysis was found to give values for in vitro protein binding in plasma equivalent to those determined by ultrafiltration. Microdialysis offers advantages in terms of maintaining equilibria and experimental versatility. Microdialysis sampling also provides potential use for in vivo determinations of protein binding.