High performance liquid chromatographic (HPLC) and enzyme immunoassay (EMIT®) methods for quantitating theophylline concentrations in serum were evaluated. For both methods, standard curves were linear over the therapeutic range of serum concentrations. Precision was acceptable, with coefficients of variation being less than 9%. The HPLC assay was slightly, but not significantly, more precise. No interference was noted in either method by caffeine, theobromine, diphylline, 8-chlorotheophylline, or by metabolites such as 1-methylxanthine, and 3-methylxanthine, or by potentially interfering compounds such as urea and uric acid. The two methods correlated well, with a correlation coefficient of 0.98%. The enzyme immunoassay was superior in terms of lower costs, greater ease of performance, and potential for automation.