Questioning is a critical strategy for science teachers to scaffold students' exploration and knowledge construction in inquiry-oriented science teaching. In science teacher preparation, open-ended questions asked by teachers are advocated as an advantageous strategy to prompt student thinking. However, insufficient attention has been cast on how science content knowledge embedded in teacher questions contributes to students' conceptual understanding. Pre-Service Teachers (PST) may formulate a mindset of hands-off inquiry teaching where students could achieve a learning objective by articulating their thoughts without guidance from teachers. In addition, existing methods for the assessment of questioning are mainly qualitative via discourse analysis from limited scenarios, which may yield biased inferences of a teacher's competency in questioning. Besides, qualitative methods are unwieldy for large-scale analyses due to the complexity of synthesizing discoursal information. In this study, we designed a written instrument for quantitative assessment of PSTs' pedagogical content knowledge of questioning. We thoroughly introduced the free-response and multiple-choice versions of this instrument and applied it with 108 PSTs. The findings supported the validity and reliability of this instrument. As suggested by this instrument, the participating PSTs were aware of the importance of questioning in inquiry teaching. However, the PSTs' difficulties with science content knowledge and knowledge of students' understanding might impede them from determining effective guiding questions to scaffold student learning. Finally, we discussed the potential of this instrument in preparing PSTs' questioning skills.