Computational thinking has become an integral component of curricula worldwide, necessitating teachers to develop this competence in their students. To effectively meet these curricular requirements, teachers themselves need a solid foundation of computational thinking content knowledge, which refers to the understanding and skills they possess in this area. However, despite widespread recognition of this need, few studies have rigorously examined teachers’ content knowledge in this domain. Addressing this gap requires the development of high-quality measurement tools. This study details the development of an instrument, created as part of the International Computer and Information Literacy Study (ICILS) 2023 in Flanders, to measure lower secondary school teachers’ computational thinking content knowledge in a valid and reliable way. The article first outlines the construction process of the instrument, which involved close collaboration with experts in the field and drew upon the framework of Fraillon and colleagues (2023). Following this, the instrument’s psychometric properties are presented, which include both item-level and overall instrument characteristics. These properties were evaluated using data from a sample of 352 participants, applying both Classical Test Theory and Item Response Theory. The final tool consists of 16 multiple-choice and short constructed response questions. The results indicate favorable item and overall instrument characteristics, thereby affirming its potential to measure the intended construct in a valid and accurate way.