(1) Background. The aim of the present protocol is to assess whether self-perception of tooth colour and facial attractiveness correlates with gender and mood state. (2) Study protocol. The protocol foresees a cross-sectional clinical study, developed by Sapienza University of Rome, Italy, Department of Oral and MaxilloFacial Sciences, and 15 UNID (Unione Nazionale Igienisti Dentali-National Union of Dental Hygienists)-affiliated clinical centers in Italy. The protocol consists of a clinical visit, during which photographic documentation of the face and smile is collected with spectrophotometric evaluation of tooth colour. During the visit, two validated questionnaires are filled in. The first one is filled by the operator for the collection of data on: i) patient’s face and smile colorimetric characteristics; ii) patient’s and operator’s evaluations of the dental colour and smile attractiveness. The second one is completed by the patient for the assessment of his/her mood state. (3) Conclusions. This protocol highlights the importance in aesthetic dentistry of a gender-specific approach and the limitations of gender-neutral models, revealing the existing gender differences in aesthetic self-perception. In addition, the colour-matching relationships between facial and dental colour characteristics will be explored. This approach improves the accuracy and personalization of aesthetic assessments in dentistry, by addressing personalized and gender-specific needs. A gender-inclusive methodology that takes a more nuanced and culturally aware approach to aesthetic dentistry is a useful adjunct to modern clinical practices.