An improved method has been developed for the determination of acetylsalicylic acid, salicylic acid, gentisic acid, and salicyluric acid in plasma and urine of rabbits and man. Samples are extracted with dichloromethane containing mephenytoin as an internal standard, the solvent is evaporated under reduced pressure, the residue reconstituted and analyzed by high-performance liquid chromatography. Extraction efficiencies, linearity and assay precision were determined. This method has been applied to human bioavailability studies and the data are presented.