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Background: ε‐Poly‐L‐lysine (PLL) is a cationic polymer consisting of 25 to 35 L‐lysine residues that adheres to the surface of skin as well as hair. However, the properties of PLL regarding its adhesion to the skin remain to be elucidated. In this study, we examined the staining of stratum corneum (SC) with fluorescence‐labeled PLL and explored its relationship with skin condition. Materials and methods: Alexa Fluor 488‐labeled PLL (AF‐PLL) was reacted with tape‐stripped stratum corneum (SC), and the staining properties were monitored by fluorescence microscopy. Clinical study was performed by measuring the water content of the cheek SC and transepidermal water loss (TEWL), and the tape‐stripped SC was subjected to staining with AF‐PLL. Results: AF‐PLL staining of the SC was inhibited at acidic pH or by the addition of high concentration of salt solution, suggesting the involvement of ionic interaction between PLL and the SC, at least in part. The AF‐PLL staining was inhibited by unlabeled PLL or various alkyl amines, but not by L‐lysine monomer. AF‐PLL staining was observed inside the corneocytes as well as surrounding cornified envelope. Clinical study revealed that AF‐PLL staining intensity of the SC was negatively correlated with its water content and positively correlated with its TEWL. Conclusion: PLL can efficiently adhere to SC and AF‐PLL staining of SC can be applied to evaluate skin conditions. [ABSTRACT FROM AUTHOR]