Teachers' practical knowledge is closely related to teaching practice. The purpose of this research was to develop an instrument for assessing upper-secondary school science teachers' perceived practical knowledge about practical work. The development of this instrument was based on five components in the conceptual framework of PCK about practical work: orientations of practical work, knowledge of curriculum materials in practical work, knowledge of students in practical work, knowledge of educational strategies for practical work, and knowledge of assessment in practical work. After the questionnaire was developed, 636 Chinese upper-secondary school science teachers participated in this research voluntarily. The results from exploratory factor analysis, correlation analysis and confirmatory factor analysis provided sufficient evidence for the construct validity of instrument. The high Cronbach coefficient indicated that the instrument had good internal consistency reliability. Finally, an instrument with six factors and 25 items was documented. The instrument would benefit science teacher educators and researchers.