Abstract: The sample was ultrasonically extracted with methanol for 15 min, after centrifugation, the supernatant obtained was used for HPLC determination. Agilent Extend C18 column (250 mm×4.6 mm, 5μm) was used as chromatographic column, and mixture of methanol and acetic acid (2+98) solution, mixed in volumic ratio of 90 to 10, was used as mobile phase. Diode array detector and the wavelength of 355 nm was adopted in the determination. The positive sample was confirmed by MS. Ion pairs used for qualitative analysis were m/z 299/255 for tretinoin and m/z 299/199 for isotretinoin. Linear relationships between values of peak area and mass concentration of tretinoin and isotretinoin were obtained in the same range of 1.00-100 mg·L-1, with same value of limit of detection (3S/N) of 0.1 mg·L-1. Tests for recovery and precision were made by addition of standard solution in 2 different concentration levels to 3 kinds of cosmetic samples, values of recovery obtained were in the ranges of 97.2%-105% for tretinoin and 97.2%-111% for iso-tretinoin with RSD′s (n=6) less than 2.5%.