Seven pairs of diastereoisomers of podophyllum lignans at 2-position, including two pairs of spin-labeled compounds, were separated by micellar electrokinetic chromatography with 5 mM sodium tetraborate-20 mM NaH2PO4-120 mM sodium dodecylsulfate-30% (v/v) 2-propanol (pH 6.5-7) within 35 min. The migration behaviors of the compounds with different types and concentrations of organic modifiers were studied. The method can be used to identify the purification of the lignans, monitor the 2-H configuration transformation, and determine the 2-H configuration of the spin-labeled derivatives.