Plants are traditionally used worldwide to fight diseases. The World Health Organization considers these traditional practices as potential sources of new treatments and encourages their study. Deciphering the pharmacological activity of herbal preparations involves several challenges, including the in-depth characterization of their chemical composition. This thesis was based on reverse pharmacology, an approach dedicated to the study of traditional practices and aimed at validating their clinical efficacy from the early stages of the research process. Thus, in this thesis, several medicinal plants or multiherb formulae studied in clinics were selected, and different projects were performed simultaneously. They ranged from studying the possible modes of action of plant constituents in vivo to permeation and biotransformation of extract constituents to predict bioavailability. The analytical and metabolomic strategies developed in this holistic context have demonstrated their ability to characterize complex plant extracts in-depth and have been used to guide advanced pharmacological evaluation.