In recent years, there has been a growing interest in studying the knowledge that mathematics teachers require in order for their teaching to be effective. However, only a few studies have focused on the design and application of instruments that are capable of exploring different aspects of teachers' didactic-mathematical knowledge about specific topics. This article reports the results obtained following the application of a questionnaire designed specifically to assess certain key features of prospective, higher secondary-education teachers' knowledge of the derivative. The questionnaire was constructed using a theoretical model of mathematical knowledge for teaching based on the onto-semiotic approach to mathematical knowledge.