Objectives: To develop and validate a questionnaire on dental students' self-efficacy with tooth removal, suitable for measuring the effectiveness of training methods. Methods: To prepare and validate this questionnaire, we used the Association of Medical Education in Europe (AMEE) stepwise guide for developing questionnaires for educational research. In the validation process, our study group conducted two pilot studies, the first for an exploratory factor analysis and the second for a confirmatory factor analysis. In addition, the questionnaire was tested for convergence with the neuroticism subscale of the NEO-Personality Inventory. Results: After an exploratory factor analysis, which used a total of 137 responses on 33 items, 15 items were left for confirmatory factor analysis. A total of 118 responses were available for the confirmatory factor analysis. Model fitness was tested using tests for exact fitness and fit indices such as the goodness of fit index (GFI), root mean square error of approximation (RMSEA) and standardised root mean squared residual (SRMR). An acceptable fit was found for 11 items divided over three factors: ‘self-perceived skill’, ‘tension’ and ‘dedication’. These 11 items did not converge with the neuroticism scale. Conclusion: This study showed the development steps and initial validation of a psychometric instrument, the Amsterdam Self-Efficacy Scale for Tooth Removal (ASES-TR), consisting of 11 items for testing dental students' self-efficacy in performing tooth removal procedures.