We used this framework to explore the results from antiparasitic screening and metabolomic profiling of 1,600 plant extracts, and anticipated two putative structural scaffolds that could be responsible for the anti-T. cruzi bioactivities observed, namely rotenoids and quinolones . Among the eight extracts active against T. cruzi, the activity of one of them (Melochia umbellata (Houtt.) Stapf stems) could be explained by the presence of quinolone derivatives (query). Due to the extensive work already done on the anti-T.cruzi activity of this scaffold, it was not deemed necessary to carry out phytochemical work on this extract . For six other active extracts, the activity was linked to rotenoid derivatives, particularly deguelin and rotenone. Biological evaluation of the commercial standards of both compounds showed nanomolar IC 50 against the amastigote form of the parasite without toxicity to the host cell . Since, to the best of our knowledge, the anti-T. cruzi activity of rotenoids had not been reported, we went on to investigate the phytochemistry of the six rotenoid-containing active extracts. We used an optimized single-step chromatography isolation workflow to efficiently and rapidly generate a library of rotenoid and related isoflavone analogs and to test them in vitro. This approach enabled an efficient translation from metabolite profiling results to targeted isolation of selected compounds and assessment of their in vitro bioactivity.