Research on teachers' professionalism and professional development has increased in the last two decades. A main focus of this line of research has been the cognitive component of teacher professionalism, i.e., professional knowledge. Most of the previous studies on teacher knowledge--such as the Learning Mathematics for Teaching (LMT) (Hill et al. 2004), the Professional Competence of Teachers, Cognitively Activating Instruction, and Development of Students' Mathematical Literacy (COACTIV) (Baumert et al. 2010), and the Mathematics Teaching in the 21st Century (MT21) (Schmidt et al. 2007) studies--have been conducted in the field of mathematics teachers' pedagogical content knowledge (PCK) and content knowledge (CK). There have been few comparable studies conducted with science teachers, especially biology teachers. To fill the gap, this study examines the development and use of instruments to measure biology teachers' CK and PCK. In particular, this study describes a method to develop reliable, objective, and valid instruments measuring teachers' CK and PCK in four steps by the use of empirical data of students. Additionally, the study explores whether CK and PCK might be measured as separate knowledge categories by using a paper-and-pencil test. This paper presents a theoretical model that guides test development and provides steps to develop and validate the instruments. Details are also provided regarding the computation of the Rasch scale score measures for 158 biology teachers. The results indicate that the instruments measured teachers' CK and PCK in an objective, valid, and reliable way. This suggests that the new instruments can be used in combination with classroom observations to examine teaching quality and further its relation to student learning.