Cystic fibrosis is a very rare and fatal desease. The most dangerous complication is chronic pulmonary desease from which almost every patient get sick. Duration and quality of life depends on severity of the pulmonary desease. Mucus that lines the airways becomes thick and sticky what then leads to the infections and shortness of breath. Drugs for treatment of cystic fibrosis are called “orphan drugs” because it is not profitable for the pharmaceutical industry to develop and sell drugs for treatment of only small percentage of population. Because of that it is very important to conduct clinical testing of new orphan drugs, so we can facilitate treatment of rare deseases. The aim of this study was to compare two extraction techniques: ultrasonic extraction and Quechers extraction. Sample of rat chow was homogenized and dissolved in solvent methanol:water=50:50, v/v %. Different extraction time (40 and 100 minutes) were investigated and ultrasonic extraction, with duration time 100 minutes, was more effective because more pirfenidone was extracted. First extraction method was performed using ultrasonic bath while the other one was a QuEChERS method. QuEChERS was made with QuEChERS equipment and by the scheme for extracting sample with fats because in rat chow there are fats that would make trouble when analysing them with an HPLC. After the analysis of the sample by LC/DAD/MS method, using Agilent 1100 Series LC/MSD trap system, ultrasonic extraction in duration of 100 minutes is selected as the better method for isolation pirfenidone from a rat chow. Despite all the advantages that QuEChERS has, e.g. simplicity, rapidity, cheapness, safety and possibility to extract more than 250 components at once, ultrasonic extraction has extracted bigger amount of pirfenidone. The difference in the time of extraction was not significant. Ultrasonic extraction lasted for 100 minutes and QuEChERS for 70 minutes. Optimized extraction conditions were successfully applied for analysis of pirfenidone in rat chow.