A study of the simultaneous separation and determination of selected polyphenols (rutin, narirutin, naringin, hesperidin, neohesperidin, quercetin, naringenin, kaempferol and hesperetin) with reported effects in the treatment of depression and cardiac and neurodegenerative diseases was performed. An RP-ultrahigh-performance liquid chromatography (UHPLC)-ultraviolet method for analyte separation and determination was successfully developed and validated for a ZORBAX Eclipse Plus C18 RRHD analytical column. Separation was carried out in gradient elution mode with acetonitrile and water modified with 0.05% trifluoroacetic acid. In the selected working range, the method linearity was satisfactory, with coefficients of determination >0.99. The precision and accuracy did not exceed the acceptable limits of 15%. The method was used to compare 16 different SPE sorbents for medicinal plant sample preparation in terms of analyte recoveries and matrix purification. The analysis of real samples was carried out for Menthae piperitae (predominant analyte was rutin), Hypericum perforatum (predominant analyte was rutin), Salvia officinalis (predominant analyte was kaempferol) and their derived products, enabling a comparison of different plant materials. Additional confirmation by UPHLC coupled with tandem mass spectrometry was performed. For the chiral aglycones naringenin and hesperetin, the determination of individual enantiomers was also performed with a Chiralpak AD-3R analytical column.