This article reports the development and application of two instruments for assessing science teachers’ pedagogical knowledge in the context of teaching higher-order thinking: a Likert-type research instrument, and an instrument that analyzes classroom observations. The rationale for developing these instruments and their main categories is described. One hundred and fifty Israeli science teachers replied to the Likert-type questionnaire. Results show that biology teachers gained a significantly higher score than either physics or chemistry teachers, that junior high school teachers scored significantly higher than high school teachers, and that a significant negative correlation was found between final scores and teaching experience. Participants in the classroom observation study were 14 teachers who attended a one-year professional development course for teaching higher-order thinking. The instrument was sensitive in detecting progress in teachers’ pedagogical knowledge in several categories, such as: Frequency of tasks that required higher-order thinking; The variety of thinking strategies that teachers addressed during their lessons; Engagement of students in metacognitive thinking; and Using the “language of thinking” in class. The implications of the findings for research and practice are described.