A pressurized liquid extraction and on-line SPE-HPLC method was developed for simultaneous determination of six polyynes, including falcarindiol, oplopandiol, (11S,16S,9Z)-9,17-octadecadiene-12,14-diyne-1,11,16-triol,1-acetate, oplopandiol acetate, oplopantriol A, oplopantriol B, and one polyene, (S,E)-nerolidol, in Oplopanax horridus and Oplpanax elatus. The analysis was conducted on a Grace Prevail C column (3μm, 7mm×33mm) with gradient elution of acetonitrile and water after the sample loaded and washed with 42%ACN in 0.3min on a phenomenex Strata-X on-line Extraction Cartridge SPE column (2.5μm, 2.0×20mm). All calibration curves of seven analytes showed good linearity within the test ranges. The validated method was successfully applied to quantify six polyynes and one polyene in two species of Oplopanax, O. horridus and O. elatus. © 2010 Elsevier B.V.