A mass fragmentographic method of high accuracy for determination of serum urea is described. A fixed amount of [^15N₂]urea is added to a fixed amount of serum, then the urea is converted into 5,5-diallyl barbituric acid by coupling with diallyl malonic acid diethyl ester. The barbiturate is then transferred from an alkaline water phase into an organic phase containing methyl iodine by ion-pair extraction using tetrabutyl ammonium as the positive counterion. The amount of urea is determined from the ratio between the recordings at m/e 236 and m/e 238 obtained after analysis with a combined gas chromatograph-mass spectrometer equipped with an MID-unit (multiple-ion detector). The two ions used correspond to the molecular peak in the mass spectrum of the methyl derivative of unlabeled and labeled 5,5-diallyl barbituic acid, respectively. The relative standard deviation of the method was 3.6%. A comparison between the mass fragmentographic method and a routine method for determination of serum urea based on the urease-Berthelot reaction gave a high correlation (r = 0.99) and a regression coefficient of 0.95.