A gradient elution HPLC method for analysis of propranolol and three basic metabolites in bile is presented. Before or after hydrolysis with beta-glucuronidase, acetonitrile is added to the bile sample, the resulting mixture is centrifuged at high speed, a portion of the supernatant is diluted with buffer and an aliquot is injected onto the HPLC column. The method utilizes gradient elution, reverse phase chromatography with fluorescence detection, and is sufficiently sensitive to quantitate 1.25 mcg/ml of compound using 50 μl to 100 μl of bile sample.