Atopic dermatitis (AD) is a common skin disease characterized by chronic inflammation and itchiness. Although skin barrier dysfunction and immune abnormalities are thought to contribute to the development of AD, the precise pathogenic mechanism remains to be elucidated. We have developed a unique, diet-induced AD mouse model based on the findings that deficiencies of certain polyunsaturated fatty acids and starches cause AD-like symptoms in hairless mice. Here, we present a protocol and tips for establishing an AD mouse model using a custom diet modified from a widely used standard diet (AIN-76A Rodent Diet). We also describe methods for evaluating skin barrier dysfunction and analyzing itch-related scratching behavior. This model can be used not only to investigate the complex pathogenic mechanism of human AD but also to study the puzzling relationship between nutrition and AD development.