Research in science education has recognized the importance of pedagogical content knowledge (PCK) and its relationship to practical work. Although efforts have been made to characterize this relationship accurately, research in this field still needs to be completed. This article shows an instrument’s design and validation process that aims to determine the perception of PCK elements and their incidence in the design, implementation, and evaluation of practical work in science teachers. The questionnaire was subjected to content validation by a panel of experts and a pilot study to evaluate its internal consistency through Cronbach’s alpha. The construction and validation yielded an instrument of 30 items grouped into five categories: orientations toward science teaching, knowledge of the curriculum, knowledge of assessment, knowledge of students’ understanding of science, and knowledge of instructional strategies. Reliability yielded a Cronbach’s alpha value of 0.881, showing the development of a consistent and reliable instrument.