A simple HPLC method was developed and validated for the determination of uric acid (UA), xanthine (X) and hypoxanthine (HX) concentrations in human serum to support pharmacodynamic (PD) studies of a novel xanthine oxidase inhibitor during its clinical development. Serum proteins were removed by ultrafiltration. The hydrophilic analytes and the I.S. were eluted by 100% aqueous phosphate buffer mobile phase. The hydrophobic matrix components (late peaks) were eluted with a step gradient of a higher organic mobile phase. Validation on linearity, sensitivity, precision, accuracy, stability, and robustness of the method for PD biomarkers (UA, X, and HX) was carried out in a similar manner to that for pharmacokinetic (PK) data where applicable. Issues of selectivity for endogenous biomarker analytes and individual concentration variations were addressed during method validation. Standards were prepared in analyte-free phosphate buffer. Quality control samples were prepared in control serum from individuals not dosed with the xanthine oxidase inhibitor. The method was simple and robust with good accuracy and precision for the measurement of serum UA, X, and HX concentrations.