Idiopathic pulmonary fibrosis is a rare, chronic and serious disease which leads to lung volume diminishment and progressive lung insufficiency due to the fibrosis of the interstitium of the lungs. The only drug approved for treating mild to moderate lung fibrosis in adults is pirfenidone. Specific and sensitive methods must be used in order to monitor the therapeutic effect of the drug and its metabolites in biological samples. The point of this thesis was to evaluate the time of retention of pirfenidone by changing the ratio of the solvents used for the mobile phase. The mobile phase consisted of purified water, acetonitril and formic acid. Different ratios within the mobile phase were used in order to shorten the time needed to perform the high-performance liquid chromatography and mass spectrometer analysis of pirfenidone in biological samples. Two columns, Symmetry C18 and Xbridge C18 were tested prior to the analysis. Even though there was no significant difference in the selectivity of the above mentioned columns, Xbridge C18 was chosen because it was shorter and it gave shorter analysis time. The Xbridge column was used to choose the optimum mobile phase consisting of water, acetonitril and formic acid for the final evaluation. Due to satisfactory time of analysis and symmetrical chromatographic peaks , mobile phase with the following solvent ratios was chosen: water: acetonitril: formic acid = 60:40:1. Mobile phase flow was set to 0,4 ml/min and the wave length of the DAD detector was set to225 nm. The influence of water in the mobile phase on the retention time, as well as the width and height of the peak of pirfenidone was also tested. Furthermore, the fragments of the mass spectre of pirfenidone were evaluated afterwards. Since the newly developed method is expected to be used for the analysis of pirfenidone in biological samples, it was neccessary to evaluate the selectivity of the method by using the DAD detector and mass spectrometer in the analysis of a serume containing 0,1 milligrams of pirfenidone per millimeter. The selectivity of the mass spectrometer was more satisfactory, as expected. Further tests shall be concentrated on determining pirfenidone and its metabolites in biological samples because, in order to monitor the therapeutic effect of pirfenidone, one must also monitor the concentration of its metabolites as well.