Aim: The objective of this study was to evaluate the rapid equilibrium dialysis (RED) device in protein binding assays in diluted protein matrices and to improve the accuracy of the unbound fraction (fu) measurement. Methodology: Protein binding assays of drug compounds in bovine serum albumin solutions and human plasma with different folds of dilution were performed using the RED device with and without preconditioning of the dialysis membrane inserts, and the results were compared with those using other approaches in this study. Results & conclusion: Preconditioning of the RED membrane inserts improved the fu data accuracy of drug-protein binding assay in matrices with relatively low protein contents and such impact could be compound dependent.