The determination of vitamin D₂ isomers in mixtures was investigated in order to use in kinetic experiments on their isomerization with acidic reagents. Vitamin D₂, pre-D₂, 5, 6-trans-D₂, iso-D₂, T₂ and iso-T₂, thought to exist in the reaction mixtures, were chosen as standard compounds of vitamin D₂ isomers. When a mixture of the standard compounds was ap-plied to the GLC using OV-17 as a stationary phase after trimethylsily-lation, the separation of vitamin D₂ from pre-D₂ and of T₂ from iso-T₂ were impossible while the two groups and the other compounds were separated one another (Both vitamin D₂ and pre-D₂ were quantitatively isomerized into the thermal cyclized compounds “pyro- and isopyro-D₂” in the same manner.). Therefore, it was possible to determine the compounds by the GLC when the coexistences of vitamin D₂ with pre-D₂ and of T₂ with iso-T₂ were simultaneously unidentified, whereas pre-treatment was thought necessary to separate them when their coexistence was identified. When a mixture of the standard compounds was applied to the TLC using Kieselgel GF₂₅₄ as an adsorbent and benzene-acetone (95:5) as a developing solvent, vitamin D₂ from pre-D₂ and T₂ from iso-T₂ were separated each other. From these results, a method based on the TLC and GLC was proposed for the determination of vitamin D₂ isomers in mixtures.