A simple reversed phase HPLC method with UV detection in isocratic conditions was developed and validated for the simultaneous determination of hypoxanthine, xanthine and uric acid levels in human plasma and serum. One analysis run takes 6.5 min including a short organic mobile phase gradient for column regeneration. Concentrations of uric acid, xanthine and hypoxanthine in plasma and serum samples were highly comparable. However, hypoxanthine levels were increased in serum compared to plasma samples due to a prolonged time between serum and blood elements separation. The method was validated for linearity, precision, accuracy, sensitivity and robustness in a similar manner to that for pharmacokinetic data and it is appropriate for physiological and pathophysiological levels of all analytes. The stability of stock standard solutions was verified using spectrophotometric analysis in different conditions. The method is simple and robust with a good precision for the measurement of hypoxanthine, xanthine and uric acid in human plasma and serum.