A novel stratum corneum substitute (SCS) has been developed, and the fundamental mechanism of the dehydration process has been studied using the SCS. After washing with cleansers which contain surfactants, our skin "feels" dehydrated (or hydrated). Although many studies have focused on the effect of surfactants on the regulation of the water loss by the lipid bilayers in the stratum corneum (SC) for a long timescale or at equilibrium, only few studies have focused on the acute effect of the surfactant interaction on dehydration. In addition, the interaction between the surfactant and keratin has been often underappreciated compared to lipid bilayers although keratin is the major nonaqueous component of the SC. Here, we have developed novel SCS models, nonkeratinized (lipid only) and keratinized, to study the effect of keratin hydrolysates on the dehydration rate. We have confirmed that the lipid organizational structure of the SCS was similar to that of the human SC using X-ray scattering. We have revealed that keratin hydrolysates play a significant role in the dehydration rate, accelerating the rate for the short term. We have also demonstrated that the effect of surfactants on dehydration is more pronounced for keratinized samples than that for the nonkeratinized sample. However, the dehydration rate for the nonkeratinized SCS with the surfactant became faster than the that for the keratinized SCS after the 20 min evaporation process, suggesting that the water binding sites of keratin hydrolysates slowed down evaporation, while the surfactant interacting with the lipids accelerated the water loss. Lastly, the study demonstrated that the SCS model can be a great platform to test macroscopic properties and analyze the underlying mechanism at the molecular level for various chemicals.