The lack of standardised assessment of evolutionary knowledge and acceptance of evolution across Europe makes comparisons between studies difficult. The Evolution Education Questionnaire on Acceptance and Knowledge (EEQ) was constructed to measure attitudes and understanding across Europe and beyond. We aimed to compile a brief instrument to allow for easy application in school and university. The target group of the EEQ was freshman university students who had just finished their secondary education. However, several components of the questionnaire were developed and validated for additional target groups. Therefore, this questionnaire may, in addition, be suitable for students in secondary school, in-service teachers as well as the general public.This method report describes the contents and application of the EEQ and provides information on survey conduction, data preparation, analyses and interpretation of results to serve as a standardised and ready-to-use protocol to measure the acceptance of and knowledge about evolution in a local, national or international context. To allow for sampling in different European countries, we present the EEQ in 23 European languages.