UHPLC in combination with electrospray ionization mass spectrometric detection in the positive ion mode has been used successfully to screen the yohimbine content in food supplements. The chromatographic behaviour of yohimbine is examined using Accela UHPLC with mass spectral detection HRMS “Q-Exative” with H-ESI-interface (“Thermo Fisher Scientific”, Waltham, MA, USA) and columns “Kynetex” RP C8 50 × 3 mm × 2.6 μm type “core-shell” and “Poroshell” RP CE18 150 × 3 mm × 2.7 mm. We have used mobile phase containing: acetonitrile, methanol water, ammonium formate. We have made series of chromatograms in positive mode of ionization and in negative mode of ionization. We have found that the ionization in positive mode offers a significantly higher sensitivity and also higher selectivity than the negative ion mode. This analytical method could be successfully applied for analytical control of food supplements containing yohimbine.