A gradient high performance liquid chromatographic method has been developed for the determination of 4-methylumbelliferone (4MU), 4-methylumbelliferyl sulfate (4MUS), and 4-methylumbelliferyl β-D-glucuronide (4MUG) in dog plasma and William's E media. Samples containing the internal standard, umbelliferone were prepared for analysis by precipitation with acetonitrile prior to injection onto a 150 x 2.1mm C18 Hypersil reversed-phase column. The compounds were eluted in less than 7 minutes with a fast linear gradient from 9.5% acetonitrile to 32% acetonitrile in pH 4.5 acetate buffer with a flow rate of 0.35 mL/min. All compounds were detected at 314 nm. Total sample cycle time was 10 minutes. Intra-run (n=5) and inter-run (n=18) precision was less than 15% relative standard deviation across the entire calibration range. The lower limit of quantitiation with a 50 μL sample size was 2.5 pM.