Abstract: The sample of skin-care cosmetic was extracted with 95% (volume fraction) methanol solution containing 0.1 mol·L^-1 ammonium acetate, and then the extract was filtered on filter membrane. LC-MS/MS was applied to the determination of 5 palmitoyl polypeptides in the filtrate. LIChrospher^® 60RP-select B chromatographic column was used as stationary phase, and the mixture of 50 mmol·L^-1 ammonium acetate solution containing 0.3% (volume fraction) acetic acid and acetonitrile mixed in different ratios was used as mobile phase in gradient elution. ESI⁺, ESI^- and selected reaction monitor mode were adopted in MS/MS. Linear relationships between values of peak area and mass concentration of 5 palmitoyl polypeptides were kept in the same range of 2.0-200.0 μg·L^-1, with the same lower limits of determination (10S/N) of 2.0 μg·L^-1. On the base of blank sample, test for recovery was made by standard addition method; values of recovery found were in the range of 89.4%-111%, with RSDs (n=6) of determined values in the range of 1.1%-10%.