Five new (1-5) and four known (6-9) C14-oxygenated 1alpha-hydroxy-11(13)-pseudoguaien-6beta,12-olides with potent inhibition of hepatitis C virus (HCV) replication were obtained from Parthenium hispitum via high-throughput natural product chemistry methods. A semipreparative HPLC system was used to purify these compounds. The miniaturization of the structure elucidation and dereplication for the mass-limited samples were performed primarily utilizing a capillary-scale NMR probe. Compounds 2-4 were found to possess in vitro anti-HCV activity in the subgenomic HCV replicon system containing luciferase reporter with significant inhibition above 90% at 2 microM concentration.