This study describes the development and application of a three-tier test as a valid and reliable tool in diagnosing students' misconceptions regarding some basic concepts about carbohydrates. The test was administrated to students of the Pharmacy Department at the University of Bijeljina (Serb Republic). The results denoted construct and content validity of the instrument and its high reliability. Considering the results entirely, it was determined that the presence of a third tier in a task significantly affected the number of identified misconceptions and the assessment of scientific knowledge. These findings suggest that certain incorrect responses should be attributed to the lack of knowledge rather than to misconceptions, and likewise, some correct responses should be attributed to the lucky guess rather than to scientific knowledge, which implies a higher credibility of the results obtained. In this study, several misconceptions about carbohydrates were revealed. As there are only a few studies examining students' misconceptions in chemistry using three-tier tests available in literature, future research should focus on the further implementation of three-tier tests as a powerful tool for simple detection of misconceptions.