Abstract: OBJECTIVE To develop a method for related substances determination of isotretinoin by HPLC. METHODS NUCLEOSIL 100-3 C₁₈ column (4.6 mm×150 mm, 3 μm) was used. The mobile phase was consisted of methanol-water-glacial acetic acid(770:225:5) with a flow rate of 1.0 mL·min^-1. The column temperature was 25℃, and detection wavelength was 355 nm. RESULTS Chromatographic peaks of isotretinoin and impurity H, I, tretinoin, degradation impurities were separated. Linear ranges of isotretinoin, impurity H, I and tretinoin were 0.000 545 5-21.82 μg·mL^-1(r=0.999 9), 0.002 856-7.14 μg·mL^-1 (r=0.999 0), 0.002 789-6.97 μg·mL^-1(r=0.999 1), 0.017 07-22.76 μg·mL^-1(r=0.999 6), respectively; LODs were 0.27, 0.60, 0.65, 5.50 ng·mL^-1; LOQs were 0.55, 2.85, 2.80, 17.00 ng·mL^-1. Average recoveries of impurity H, I and tretinoin were 101.57%, 102.02%, 101.03%, and RSDs were 0.5%, 0.8%, 1.5%. CONCLUSION The HPLC method is accurate, specific and reliable for determination of related substances of isotretinoin.