Abstract <p>This study presents a rapid and highly sensitive approach for determining seven major bioactive lignans—matairesinol, nortrachelogenin, pinoresinol, hydroxymatairesinol, secoisolariciresinol, lyoniside, and ssioriside—in plant extracts using supercritical fluid chromatography–tandem mass spectrometry in the multiple reaction monitoring mode. Analyte ionization was studied as a function of the ion source, with comparing electrospray ionization with atmospheric pressure chemical ionization. Lignan retention on commonly used SFC stationary phases was studied and the best results were achieved using the HSS C18 SB phase, with separation governed primarily by interactions with residual silanol groups. Optimization of chromatographic separation and mass-spectrometric detection parameters enabled the simultaneous quantification of all analytes within a 10-min run, with limits of detection ranging from 0.25 to 3.13 μg L^–1. The developed approach was validated and tested by analyzing extracts from aboveground parts of berry plants and compression wood of coniferous trees.</p>