A simple and specific reversed-phase high-performance liquid chromatographic (HPLC) assay for the determination of novobiocin levels in human plasma has been developed. The sample preparation was performed by deproteinization with methanol. Prednisone was used as an internal standard. Both novobiocin and prednisone were separated on a C8 column with a gradient elution of acidic water (pH 3.0)-methanol. The recovery of novobiocin from plasma was nearly complete. The linear range was 5-1000 microM in 0.5 ml of plasma with a minimum limit of determination of 2.25 fmol of novobiocin at 254 nm. The method has been implemented and validated in an ongoing clinical trial.