Cell biology is widely considered to be the most important subject for promoting current modern biology. This paper sheds new light on the subject with the development of a diagnostic test to investigate misconceptions among pre-service biology teachers in cell biology. In this study, we developed and validated a diagnostic test -- known as the three-tier test -- to investigate misconceptions. In developing the test, we modified the initial version of the Cell Biology Diagnosis Test (CBD-Test), comprising a two-tier construct, and then we added a certainty response index for each item. The initial CBD-test was a construct based on proportional knowledge, references, and concepts that have been identified as frequently misunderstood in cell biology. The three-tier test was then adjusted to distinguish a lack of knowledge from a misconception. Participants in this test were 204 pre-service student teachers in an undergraduate biology education program. The test highlighted the fact that most of the students shared misconceptions regarding the structure and function of cells, fundamental components of cells, material transport across the membrane, and cell division. Furthermore, the findings validate the advantages of the CBD-Test as a reliable and valid instrument to diagnose misconception on a broad range.