The separation of astaxanthins from carotenoids from freshwater amphipod, yeast, green alga and flower plant using by HPLC is described. The stereoisomers of astaxanthins were separated by means of chiral column and their structures were confirmed by circular dichroism spectra. The resolution of single stereoisomers of astaxanthin has α=1.048 and α=1.056 respectively. The future scope of chiral HPLC, including the separation of the former consistent astaxanthins from green alga and yeast is also discussed.