The permanganate oxidation method for the spectrophotometric determination of phenylbutazone has been modified and optimized to permit precise assays of phenylbutazone in small plasma samples. The assay is not affected by the presence of p-hydroxyphenylbutazone, the major metabolite of phenylbutazone. Weak bases are removed in the initial extraction procedure, and weak acids such as phenobarbital, warfarin, diphenylhydantoin, salicylic acid, and sulfadiazine do not interfere in the assay.