To evaluate drug-protein binding, a sensitive method for the determination of ibuprofen in submilliliter amounts of serum was required. A specific and highly sensitive procedure, based on benzene extraction of the acidified specimen. TLC of the benzene extract residue, fomation of the pentafluorobenzyl esters of the materials eluted from the thin-layer chromatogram, and quantification of the pentafluorobenzyl esters by GLC, was developed. Utilizing electron-capture detection, the method is sensitive to 0.1 microgram of ibuprofen/0.1 ml of serum. Statistical analyses indicated an average recovery of 97.7% with a standard deviation of +/- 7.3%. Mass spectrometric analysis, in conjunction with GLC, confirmed the specificity of the method for the intact drug. The procedure was applied successfully to drug absorption and drug-protein binding studies in humans.