Palmitoyl peptides are anti-aging agents widely used in cosmetics. This article describes the development of a LC-MS/MS analytical procedure that allows, after a liquid-liquid extraction procedure, their unambiguous detection in cosmetic formulation. MS/MS detection is shown to be specific regarding placebo formulations. Limits of quantification, linearity, accuracy and precision of the method were estimated. The results presented show that palmitoyl peptides can be thus reliably assayed. The palmitoylated pentapeptide palmitoyl-lysyl-threonyl-threonyl-lysyl-serine (pal-KTTKS) was assayed in anti-wrinkle creams using palmitoyl-glycyl-histidyl-lysine (pal-GHK) as internal standard. From the results obtained, the influence of the formulation on pal-KTTKS availability is evidenced.