The method described for the estimation of squalene involves chromatographic separation of the dehydrogenated reaction products of squalene with N-bromosuccinimide into several parts of DS 1〜6 (adsorbent : lime, developer of chromatograms : 10〜20% acetone in hexane), followed by spectrophotometric determination of DS 4 at 395 mμ. This method would be effective particularly for estimating squalene of natural source in the presence of other unsaturated polyenes. The configurations of DS l, 2 and 3 were assigned tentatively to be all-trans-, 10 or 14-monocis-hexamethyltetracosaundecaene and all-trans-hexamethyltetracosanonaene respectively.