A spectrophotometric assay procedure is described for individually measuring theophylline-7-acetic acid and theophylline in 2.0 ml of serum. Absorbance is a linear function of concentration over a range of 0.5-40 mg/liter for theophylline-7-acetic acid and 0.5-40 mg/liter for theophylline. No endogenous or exogenous compounds were found that interfere with theophylline-7-acetic acid determinations. As with other spectrophotometric theophylline assays, theobromine and, to a slight extent, phenobarbital interfere with theophylline determinations.