A high-performance liquid chromatographic (HPLC) assay first described in 1979 has been modified and revalidated for the simultaneous determination of prednisone, cortisol and prednisolone in human plasma using betamethasone as an internal standard. Revisions include: mobile phase composition; use of a precolumn, automated injector, integrator, and computer software; improved sensitivity and quantitation; thorough investigation of stability, variation, and specimen type; and inclusion of suggested quality control criteria. Plasma-based drug standards are extracted with methylene chloride and washed with sodium hydroxide followed by a water wash. After evaporation of solvent and reconstitution with mobile phase, the extracts are then injected onto a silica gel column (Zorbax SIL) for chromatography with UV absorbance at 254 nm. Calculated limits of quantitation are 10 ng/ml and limits of detection are less than 5 ng/ml. Intra- and inter-day coefficients of variation for quality control samples for all three corticosteroids are less than 11.2%. Recovery and stability data are also provided. Several drugs that may be coadministered do not interfere with the analysis.