The goal of the study was to investigate comparatively the performance of the conventional equilibrium dialysis method using artificial membranes (AED) and an alternative equilibrium dialysis method employing biological membranes of red blood cells (BED). The following criteria were employed for an assessment of the two methods: (a) mean estimate of the fraction of drug unbound in plasma, (b) precision, and (c) time required for establishing equilibrium dialysis. For this purpose, plasma protein binding data by AED and BED obtained for several compounds in our laboratory were employed. In addition, suitable results of further compounds on the plasma protein binding by AED and on the partitioning in red cell buffer and plasma systems were collected from the literature, allowing a calculation of the plasma protein binding by BED. Plasma protein binding values by AED and BED were available for a total of 22 nonelectrolytic and electrolytic compounds, including the entire possible range of binding values. Plots of the mean plasma unbound fractions as obtained by AED and BED for the compounds studied could be fitted by a straight line with slope and intercept not significantly different from unity and zero, respectively. Also, the precision of the two methods appeared to be similar. However, the times required to reach equilibrium dialysis were significantly different: With BED and AED, this time span ranged between 2 and 45 and 180 and 960 min, respectively. These results indicate that overall the BED method offers a significant advantage over the AED procedure: It is less time consuming and hence possibly more reliable.