Acylation with long-chain fatty acids is a common modification at the N-terminal glycine residues of natural proteins. In this work, we performed HPLC analysis of myristoylglycine (Myr-Gly-OH), palmitoylglycine (Pal-Gly-OH) or lauroylglycine (Lau-Gly-OH), which were produced in the hydrolysates of synthetic Myr-Gly-, Pal-Gly-, or Lau-Gly-peptides, respectively, by means of a mild acid hydrolysis in methanesulfonic acid : dioxane : water (2 : 1 : 1) at 60 °C for 12 h. Myr-Gly-OH, Pal-Gly-OH and Lau-Gly-OH were quite stable under hydrolysis conditions. These fatty acyl-Gly-OH were conveniently detectable at a 20 nmol level by direct reversed-phase HPLC. Thus, mild acid hydrolysis, followed by HPLC analysis of the hydrolysate, provides a simple method of identification of the N-terminal structure of fatty acyl-Gly-peptides.