A combinative method using overpressured layer chromatography (OPLC) and TLC bioautography against O2(•-) was developed to separate antioxidants from Glehnia littoralis roots. Through target-directed isolation by the TLC bioautographic method, seven compounds including five antioxidants were rapidly isolated by OPLC and identified as 1-linoloyl-3-palmitoylglycerol, facarindiol, panaxynol, isoimperatorin, β-sitosterol, scopoletin, and umbelliferone from G. littoralis roots. This OPLC method is a very suitable separation technique for light-sensitive polyacetylenes (panaxynol and facarindiol) with higher isolated yields compared to conventional open column chromatography. This is the first report on the separation of polyacetylenes by OPLC.