Abstract: OBJECTIVE To establish a method for the determination of tachysterol in vitamin D₂ soft capsules using two-dimensional liquid chromatography. METHODS Test wavelength: 281 nm; velocity of flow 0.5 mL·min^-1; column temperature: 40 ℃. One-dimensional chromatographic condition: Thermo Acclaim HILIC propylamide bonded silica gel column (2.1 mm×150 mm, 3 μm) was used, hexane-pentyl alcohol-isopropanol(998：0.5：1.5) as mobile phase. Two-dimensional chromatographic condition: Thermo silica column(3 mm×100 mm, 1.8 μm) was used, hexane(A)-hexane-pentyl alcohol- isopropanol(96：2：2)(B) as mobile phase, gradient elution. RESULTS In the range of 0.032 5-0.325 μg·mL^-1, tachysterol could obtain a good linear relationship(r=0.999 9), the recovery of tachysterol was 99.35%(RSD=1.4%, n=9). The relation correction factor of tachysterol was 0.047 6, the LOD was 1.6 ng·mL^-1, the LOQ was 4.8 ng·mL^-1. CONCLUSION The method conform to the requirement of impurity determination, and the content of the tachysterol in vitamin D₂ soft capsules can be accurately measured.