Background and Objective: Lipids in the stratum corneum (SC) are of major importance for the skin barrier function. Many different methods have been used for the collection of SC for the analysis of SC lipids. The objective of the present study was to validate the cyanoacrylate method for the collection of SC in relation to lipid analysis. Methods: The results of the lipid analysis (ceramide/cholesterol and ceramide profile) of SC samples obtained by the cyanoacrylate method were compared to the results of the lipid analysis of mechanically removed SC samples. The intra- and interindividual variations in lipid composition were assessed when using the cyanoacrylate method, and lipid compositions in cyanoacrylate samples and samples taken from different depths of SC were compared. Results: No statistically significant differences were found between mean values of lipids from the mechanically removed total thickness of the SC and cyanoacrylate samples. With respect to the cyanoacrylate samples, the intraindividual variation was significantly smaller than the interindividual variation, and the results did not indicate a change in lipid profile related to the depth of SC. The results clearly indicate that the cyanoacrylate method used for obtaining SC for lipid analysis is a useful and valid method for the purpose.