The purpose of this study was to develop a valid and reliable two-tier diagnostic test to assess students' undergraduates' understanding of some concepts in the context of undergraduate general chemistry course. For developing this test, firstly, content boundaries, learning objectives and alternative conceptions were identified. The first version of the test with an open ended part was applied on 68 pre-service science teachers, and qualitative analyses were made on open-ended part. According to results, both tiers were made in multiple-choice format. The test was validated by five chemistry educators and test was applied with 151 pre-service science teachers. The final version of the two-tier diagnostic test was consisted of 44 items, reliability coefficient was found to be 0.84. (C) 2015 The Authors. Published by Elsevier Ltd.