The topic of "teacher professionalism" is one of the most crucial ones in quality education research. It has a potential to generate results that could inform and hence enhance the practice in classrooms. Thus, research in this field needs reliable instruments to measure the professional knowledge of our teachers to be able to generate reliable results for our research problems. Not many instruments have been developed with regard to this topic. At the same time, an adequate validation of the instrument developed is often missing (Schilling & Hill, 2007). Hence, in a bigger project "ProwiN" (German acronym for professional knowledge of science teachers), test instruments for measuring science teachers' pedagogical, pedagogical content and content knowledge (PK, PCK, and CK) were developed for the subjects biology, chemistry and physics. The present study tested the validity of some of these items which were used to measure the pedagogical content knowledge (PCK) of biology teachers. These items focused on measuring teachers' professional knowledge by analyzing 1) teachers' "knowledge about student understanding" (or lack of understanding) of several topics in biology and 2) "knowledge about instructional strategies" like the use of models or experiments. The content validity of these instruments was examined by think-aloud interviews with American and German Biology teachers (N = 11). This study shows a high content validity for these items. Furthermore, this paper demonstrates the scope for adapting the conceptual framework of these items to measure biology teachers' PCK in other countries.