Background: Compatibility determination between active pharmaceutical ingredients (APIs) in fixed-dose combinations is an indispensable step in the elaboration. High-performance liquid chromatography provides information on possible interactions between APIs and their related interaction products. The purpose of the present study was to investigate the compatibility of potassium orotate in combination with spironolactone by a HPLC method. Material and methods: The detection was carried out using Liquid Chromatograph Agilent 1100 with UV-VIS detector and a RP-18 reversed column (250*4 mm, 5 μm), mobile phase of acetonitrile: phosphate buffer solution (pH=4.0) with the ratio 1:49 and 1:1, at flow rate 1 and 1.5 mL/min, injection volume 20 μL; potassium orotate and spironolactone substances were provided by Sigma Aldrich, USA. Results: Due to the developed method both separation and simultaneous qualitative and quantitative determination of APIs in the mechanical mixture were carried out. Spironolactone: retention time 6.9 min, concentration 98.1% (±0.21); potassium orotate: retention time 3.06 min, concentration 91.67% (±0.15). There were just well-separated symmetrical peaks of APIs and no additional peak in the chromatograms. Conclusions: There is compatibility between APIs. Further studies will be performed by other methods (DSC, FT-IF Spectrometry) to confirm the obtained result.