The purpose of this paper is to develop a "valid and reliable laboratory safety questionnaire" which could be used to identify science teachers' understanding about laboratory safety issues during their science laboratory activities. The questionnaire was developed from a literature review and prior instruments developed on laboratory safety issues. To address content validity, the questionnaire was examined by experts from the field of science education. The questionnaire consists of 36 Likert-type items related to chemicals, usage of glassware equipment, fire and electrical control, personal protection, biological hazards, and emergency. The study was carried out with 127 teachers who have experience in science laboratories and work in middle schools in Turkey. The instrument was found to be internally consistent with high reliability scores. Significance value shows that the data come from a multivariate normal distribution and are suitable for factor analysis. The factor analysis indicates that the items in the questionnaire accumulate around a single dimension named as safety issues. The results provide evidence that the instrument is valid for further implementation on a wider scale and in larger samples. The results of this study showed that the questionnaire has an appropriate scale to determine the middle school science teachers' understanding toward laboratory safety issues.