AbstractThe technology for generating high quality keratin films has recently advanced and led to their implementation in a variety of applications. As an initial step toward investigating the films as a screening tool for predicting the efficacy of potential hair dyes, CI Acid Orange 7 was applied to a set of opaque and translucent films. Overlaid time‐of‐flight secondary ion mass spectrometry images arising from protein and dye fragments revealed that dye uniformly penetrated both film types. Results also showed that the relative concentration of dye in each film complemented outcomes from ultraviolet‐visible analysis and revealed that the translucent film contained a higher dye concentration. Scanning electron microscopy analysis of film morphology suggested that the observed difference was due to the higher porosity of the opaque film, which facilitated dye desorption during the rinsing step. Consequently, the translucent film was judged to be a better substrate for screening potential new hair dyes.