Purpose: The significance of visuo-semiotic models in biology education has increased. Students have to develop visuo-semiotic skills, which could enable them to learn biology effectively. However, a lack of a universal theory of visual literacy has made it challenging to develop and assess visualization skills, including visuo-semiotic skills. The aim of the present research, therefore, was to develop an instrument for assessing visuo-semiotic reasoning in biology (VSR-b) in the context of amino acid structures. The research question guiding the research was "how could an instrument for assessing visuo-semiotic reasoning in biology be developed?" Methods: Guided by a theoretical framework, the VSR-b Test was developed using a mixedmethods approach, by first identifying VSR-b Skills through a panel of nine experts after which items were designed and validated through the same panel of experts and pilot participants (n=18). The VSR-b Test was then tested on a group of molecular biology students (n=30). Findings: Results showed satisfactory reliability and inter-item correlation. However, further research is required to corroborate findings of the present research in other contexts, with particular emphasis on assessment and development of visuo-semiotic reasoning among students. Implications for research & practice: The current research has shown that VSR-b can be understood and assessed within the context of the theoretical cognitive process of visualization. It provides teachers and researchers a starting point in understanding how learning occurs through visuo-semiotic models. Instructional and curriculum designers, therefore, can use findings of this research as a guide to support student development in biology.