Assessment literacy is a crucial aspect of teachers’ professional knowledge and relevant to fostering students’ learning. Concerning experimentation, teachers have to be able to assess student achievement when students form hypotheses, design experiments, and analyze data. Therefore, teachers need to be familiar with criteria for experimentation as well as student conceptions of experimentation. The present study modeled and measured 495 German pre-service teachers’ knowledge of what to assess regarding experimentation competences in biology. We applied an open-answer format for the measurement instrument. For modeling we used item response theory (IRT). We argue that knowledge of what to assess regarding experimentation competences is a one-dimensional construct and we provide evidence for the validity of the measurement. Furthermore, we describe qualitative findings of pre-service teachers’ knowledge of what to assess, in particular difficulties concerning the assessment of student conceptions as well as the use of scientific terms in the assessments. We discuss the findings in terms of implications for science teacher education and further research perspectives.