<p>In biology education, it is essential to systematically develop pupils' inquiry skills, with a particular focus not only on their procedural component but also on understanding their scientific nature. However, conventional tests do not provide a comprehensive view of the pupils' acquired inquiry skills. For this reason, it is necessary to explore new approaches to assess them and provide deeper insights into pupils' thinking when solving various items through scientific methods. One perspective instrument can be a four-tier test, which has become increasingly popular among researchers. Nevertheless, it has more often been applied to assess conceptual understanding of knowledge rather than inquiry skills. However, the results of our research indicate that the four-tier test represents a valid and reliable instrument that provides more objective results in contrast to the one-tier and two-tier multiple-choice tests, which significantly overestimate the pupils' mean score, and in the case of the two-tier test, also overestimate the mean frequency of pupils' misconceptions associated with the application of inquiry skills. In addition, using the four-tier test, we identified frequent misconceptions among pupils associated with skills such as designing experiments, identifying variables, and formulating research questions. These findings can contribute to preventing or eliminating these misconceptions in biology education. Overall, the results show that the four-tier test can provide more detailed information about pupils’ difficulties and problems, which can help us find more effective strategies for their development in biology education.</p>