Ultraviolet spectrophotometry is widely used for the analysis of theophylline in blood, for purposes of monitoring therapy, and for pharmacokinetic studies. Phenobarbital, a component of common oral theophylline preparations, interferes with assays now in use. The modification of the method of Schack and Waxler [J. Pharmacol. Exp. Ther. 97, 283 (1949)] presented in this paper eliminates barbiturate interference. After solvent extraction and back extraction into NaOH, the pH of the alkaline solution is adjusted from pH 13 to 10 before spectrophotometric measurement. This shifts the barbiturate absorption maximum from 255 to 240 nm, permitting accurate analysis of theophylline, the spectrum of which is unaffected by the pH change.