Pirfenidone is a novel antifibrotic, antioxidative and antiinflammatory agent registered for the therapy of idiopathic pulmonary fibrosis. In addition, it shows significant therapeutic potential in heart, kidney and liver tissue, due to which it is still under research. Its main metabolite, 5-carboxypirfenidone, is also partially responsible for the total antifibrotic action. The aim of the work was to develop a selective and sensitive method for the analysis of pirfenidone and 5-carboxypirfenidone within the matrix of a complex biological sample, right rat ventricle. In High Performance Liquid Chromatography, a Symmetry (Waters) C18 (4.6 × 150 mm, 3.5 μm) stationary phase was used, with the temperature of 28 °C. Mobile phases of various acetonitrile or ammonium formate vs. water ratios were tested, along with isocratic and gradient elution and different flow rates. Isocratic elution by a mobile phase consisting of ACN:H2O:HCOOH = 50:50:0.1% (V/V/V) at a 0.4 mL/min flow was chosen as best. The retention times of pirfenidone and 5-carboxypirfenidone under such conditions were 5.9 and 4.3 min, respectively. For the detection, DAD, FLD and MS detectors were used. DAD was applied in the preliminary HPLC method optimisation, but proved insufficiently sensitive for the analysis of complex biological samples with low analyte concentrations. FLD showed a potential in detecting both compounds, although it requires further optimisation. MS detector enabled a selective and sensitive detection in the presence of a complex matrix by using Extracted Ion Chromatograms of m/z 186 for pirfenidone and 216 for 5-carboxypirfenidone. Also, their MS2 fragmentations were studied. Paracetamol was chosen as the internal standard due to its favourable retention time (3.85 min) and the characteristics (polarity and molar mass) similar to the analytes. As extraction solvents, pure and 50% acetonitrile along with pure methanol were tested. An additional extraction of the residual precipitate with water was performed in order to check the efficiency of the aforementioned solvents for the extraction of the more polar 5-carboxypirfenidone metabolite. Pure methanol turned out to be the most favourable solvent choice for pirfenidone, while 50% acetonitrile was also satisfactory. 50% acetonitrile provided highest extraction yields for 5-carboxypirfenidone, although methanol also proved acceptable. However, pure acetonitrile was not a good extraction solvent for either of the analytes.