Cysteinyl leukotrienes cause many pathophysiological efects: bronchial hyperreactivity and bronchospasm, increased microvasculare permeability, mucous secretion. Reliable and reproducible estimation of cysteinyl leukotrienes biosynthesis is useful in detection and evaluation of diseases and also in monitoring of treatment’s efficcacy. The aim of this work was to develop, characterize and validate a new method of quantitative analysis of LTE4, and to test its practical application among patients with asthma, ischemic heart disease and healthy subjects. The method of quantitative analysis of cysteinyl leukotrienes in urine based on immunoaffinity extraction and mass spectrometry and was successfully developed and proved useful in clinical research. The method provides reliable and reproducible results of LTE4 measurement. The assay range is 6.7-2000 pg LTE4/ml of urine and the detection limit is 3.3 pg/ml. In the group of 50 healthy adults the mean uLTE4 concentration is 32.72 ± 25.10 pg/mg of creatinine. The range of reference values of LTE4 in urine is 5.69 – 119.42 pg/mg of creatinine. Among patients with ischaemic heart disease, whom percutaneous coronary intervention was performed, an increase of average 40% uLTE4 concentration within first hours after the procedure was noted. During investigation based on 110 observations of patients with coronary artery disease it was shown that zileutone decreased on average 30% the production of leukotrienes. In addition, increased systemic biosynthesis LTE4 was documented by uLTE4 excretion measurements in the group of 38 asthmatic patients. A distinct phenotype of bronchial asthma – aspirin-sensitive asthma, was found to correlate with elevated concentration of LTE4 in urine, by comparison with asthmatics who tolerated aspirin well.. Results obtained by the method were compared the commercial immunoenzymatic (ELISA) Kit. On the basis of comparison results, LC/MS method of measurement of uLTE4 was found more accurate and precise than ELISA. LC/MS method should be also considered as very specific, and very reliable.