A PEG-modified reversed migration MEEKC method was developed for simultaneous determination of six polyynes, including oplopandiol, falcarindiol, oplopandiol acetate, (11S, 16S, 9Z)-9,17-octadecadiene-12,14-diyne-1,11,16-triol, 1-acetate, oplopantriol B, and oplopantriol A, in Oplopanax horridus and Oplopanax elatus. The running buffer containing 0.8% v/v ethyl acetate, 3.8% w/v SDS, 6.6% v/v n-butanol in 20 mM phosphate buffer (pH 2.5), followed by mixing with propan-2-ol at 30% v/v and PEG-1000 at 15% w/v, was applied in the analysis. The proposed method was successfully applied to determine the six polyynes in five samples of Oplopanax horridus and one of O. elatus. The result showed that the types and amounts of polyynes present were obviously different when comparing the two herbs. Besides, the developed PEG-modified reversed MEEKC method might be suitable for the analysis of hydrophobic analytes in herbal medicines.