Teachers' pedagogical content knowledge (PCK) is highly important for effective design and implementation of school teaching. Thus, the current status, development and efficacy of this knowledge, its relationships with teaching quality parameters, and its impact on students' learning processes and success, require rigorous examination. Thoroughly validated, objective and reliable test instruments that are highly sensitive to changes in variables of proven knowledge-related relevance in teacher education are also required. Previous attempts to design such instruments for assessing science teachers' PCK have largely focused on mathematical content. Therefore, here we present an instrument (the pedagogical content knowledge in biology inventory, PCK-IBI), based on conceptualizations of teachers' professional competence, for assessing secondary school preservice biology teachers' PCK. In a series of three evaluations and refinements it was tested with samples of N = 274 and N = 432 German pre-service as well as one sample of n = 65 German pre-service and n = 35 German in-service biology teachers. Item analysis, scale analysis and empirically obtained indicators of validity suggest that the final 34-item-version of the PCK-IBI is unidimensional, provides objective test scores and enables reliable and valid registration of pre-service biology teachers' PCK. Thus, hypotheses regarding specific aspects of the model on which the PCK-IBI's construction is based on are empirically supported. The results of our study provide empirical support for the instrument's potential utility. K E Y W O R D S assessment, learning success, pedagogical content knowledge (PCK), teacher education † These authors contributed equally to this article.