Background The professional knowledge of pre-service teachers is highly important for effective and successful teaching. In recent years, many research groups have been engaged in developing simulated classroom environments to capture especially the pedagogical knowledge (PK) of pre-service teachers, neglecting the content-related facets of professional knowledge such as pedagogical content knowledge (PCK). Objectives In the present study, we describe the development of a simulated classroom environment-the Simulated Classroom Biology (SCRBio)-and provide evidence regarding its validity to assess pre-service biology teachers' action-oriented PCK in the area of evolution. Methods This study examined the evidence supporting the validity of using the SCRBio to investigate action-oriented PCK of pre-service biology teachers. The (1) evidence based on test content (expert ratings) and the (2) evidence based on relation to other variables (known-groups comparison) was obtained. We tested the SCRBio with N = 76 German pre-service biology teachers. Results and Conclusions Our results show the successfully operationalized PCK in the SCRBio through explicit allocation of specific misconceptions to each virtual student's answer and the valid measurement of pre-service biology teachers' action-oriented PCK. This results in a validated simulated classroom environment for pre-service but also in-service teachers. In the future, the SCRBio will be developed from an assessment instrument to a training tool to simulate explicit teaching situations. This allows to complement the predominantly theoretical components of university-based teacher education with practice-based simulated classroom environments.