A procedure is described to simultaneously quantitate phenolphthalein and its glucuronide metabolite from dog serum, urine and bile using high-performance liquid chromatography. The major advantages of this over pre-existing methods include direct analysis of the parent compound and glucuronide metabolite without enzymatic hydrolysis, increased sensitivity and the potential for automation of a large number of samples. Analytes were extracted from serum and urine using a combination of liquid- and solid-phase extraction methodology. Bile samples were analyzed directly after a twenty-fold dilution with mobile phase. The components plus internal standard were separated by reversed-phase high-performance liquid chromatography using step gradient elution and quantitated by the absorbance of ultraviolet light at 230 nm. Limits of detection from 1 ml of serum, 0.1 ml of urine and 0.05 ml of bile were 0.1, 0.5 and 10 microgram/ml for phenolphthalein and 0.1, 10 and 50 microgram/ml for phenolphthalein glucuronide, respectively.