Peptide medical beauty cosmetics have gradually attracted people's attention due to their excellent beauty effects. In peptide beauty cosmetics, sodium hyaluronate and excipient mannitol are usually added to stimulate collagen regeneration. The effective ratio of peptides, sodium hyaluronate, and mannitol helps with skin antioxidant and rejuvenation. Due to the much larger molecular weight of sodium hyaluronate (around 300000) compared to mannitol (182), there are significant challenges in quantitatively analyzing both components simultaneously. This application brief introduces an analytical method for simultaneously determining the content of mannitol and sodium hyaluronate in cosmetics using the Agilent 1260 Infinity III HPLC system. The performance evaluation results of the method showed that there was a good linear relationship between mannitol and sodium hyaluronate in the concentration range of 0.1-2.5 mg/mL, with linear correlation coefficients (R2) higher than 0.999; The spiked recovery rates of mannitol and sodium hyaluronate are within the range of 98.0% -101%, and the relative standard deviation (RSD) of the recovery rates from five parallel determinations is less than 0.5%. The developed method is accurate, reliable, and convenient, suitable for simultaneously determining the content of mannitol and sodium hyaluronate in cosmetics.