Inflammatory bowel diseases, including Chron disease and ulcerative colitis, are autoimmune diseases of unknown etiology characterized by chronic inflammation in digestive tract. Immunosuppressant’s are with amino salicylate, corticosteroids and biological therapy one of the major groups of medicines used in the treatment of IBD. Azathioprine and 6-thioguanine are most frequently used by immunosuppressant. These are drugs that reduce the inflammatory reaction, suppressing an over-immune response. Inflammatory bowel disease can cause folate deficiency. Low folate status may increase the risk of developing cancer, especially cancer of the colorectal. Therefore, patients should take folic acid supplement daily. We proposed the development of a new analytical method for identification of azathioprine, 6-thioguanine and folic acid in tablets and plasma control samples. The aim of this work was to develop a unique method for the determination these drugs using high-performance liquid chromatography with diode-array detector and electrospray ionization mass spectrometry. A binary gradient elution method was used as investigated compounds are structurally different compounds. All experiments were performed using Agilent Series LC/MSD Trap system. A Symmetry C18 analytical column and the mobile phase consisted of 0.1% Formic acid in methanol (eluent A) and of 0.1% Formic acid in purified water were used. The MS system was operated with electrospray ionization source in the positive ion mode. Nitrogen is used both as drying and nebulizing gas. The flow of nebulizer gas was increased because of the higher amount of water in the mobile phase. Finally, a relatively short method for determining all three analytes was proposed and the applicability of the analytical drug identification method was tested. Further studies will be focuses on the validation of the proposed method.