A bonded reversed phase, high pressure liquid chromatography (HPLC) method for serum quinidine and its major metabolite in serum, (3S)-3-hydroxyquinidine, is presented. The method employs toluene, rather than benzene for extraction of 100 microliter of serum, can utilize either absorbance or fluorescence detectors, and utilizes a column and equipment which is also suitable for theophylline analyses. Quinidine values in sera by the present method correlated well with fluorometric non-chromatographic methods using ethylene chloride (r = 0.932) or benzene-sulfuric acid (r = 0.950) for extraction of the quinidine. The comparison data suggest a therapeutic range for quinidine of 1.3--5.0 mg/liter when measured by HPLC. In over one year of routine use in a clinical chemistry laboratory, the method has proven to be rapid and precise with interassay coefficients of variation of 2.5--5.5%. No interferences with the HPLC method have yet been identified.