L-Thyroxine (L-T4), the naturally occurring thyroid hormone has been used for the treatment of thyroid dysfunctions, while D-T4 is not active in the thyroid and is used for the treatment of hypedipidemia.Therefore, a convenient and reliable enantiomer separation method has been of great interest in the biological and pharmacological research field.We demonstrated the simultaneous determination of D-T4 and L-T4 on a chiral crown ether chiral stationary phase derived from (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid in human plasma with base-line separation.This new analytical method for the determination of D-T4 and L-T4 using internal standard has been developed and validated.Validation experiments have shown that this method has good precision and accuracy as well as high linearity of 0.9987 and 0.9996 for D-T4 and L-T4 over the concentration ranges of 1-100μg/mL, respectively.This new analytical results using the chiral HPLC column may be used to investigate not only the simultaneous determination of D-T4 and L-T4 for biological samples but also the degree of conversion of each T4 enantiomer in vivo.