Ultracentrifugation methods have been widely used for the determination of the free fraction of compounds in plasma, especially for lipophilic compounds. To estimate the effect of contaminated proteins in the ''protein-free phase'' fraction, 200 mL of human plasma was separated into three layers by ultracentrifugation at 436 000g for 140 min with a table-top ultracentrifuge. Twenty microliters of the middle layer was taken as the protein-free fraction. Major contaminated proteins were analyzed by liquid chromatography/electrospray ionization/tandem mass spectrometry (LC/ESI/MS/MS) and identified as albumin, a-1-antitrypsin, a-2-HS-glycoprotein, apolipoprotein E, and apolipoprotein A-1. a-1-acid glycoprotein was not detected. Contamination of albumin was 0.13% of that in plasma. Simulation analysis demonstrated that at an actual free fraction of 1% (protein binding ratio of 99%), the extent of overestimation of free fraction was just 13% and the apparent free fraction was 1.13%. Human plasma protein binding ratios of 10 drugs estimated by this method correlated well with reported values determined by other methods, such as ultrafiltration and equilibrium dialysis, with a correlation factor of 0.98 and a slope of 0.99. Collectively, our results indicate the reliability of this micro-scale ultracentrifugation technique for the evaluation of the protein binding of drugs despite a little contamination of albumin.