A gas chromatographic/mass fragmentographic method is described which permits the determination of unconjugated pterins in urine. After the addition of 6,7-dimethylpterin as an internal standard, the acidified urine samples are purified by liquid chromatography on Dowex-50 and Dowex-1 columns. The pterins are then converted to their corresponding trimethylsilyl derivatives and the base peaks of biopterin (m/e 409), neopterin (m/e 409) and 6,7-dimethylpterin (m/e 320) are determined. The method is sensitive and specific and permits the processing of large numbers of samples. By means of this method, the urinary excretion of biopterin and neopterin from 9 healthy subjects has been determined.