The use of external representations, such as diagrams and animations, for teaching and learning in biochemistry, has rapidly increased over the past decade. Some research has shown that ERs can have a superior learning advantage over text alone, while other studies have indicated that external representations can cause and expose various visual literacy problems. The current study aimed to investigate the nature of visual literacy by identifying what visualization skills might compose optimal (expert) visual literacy in biochemistry, and to devise a means of measuring the visual literacy levels of biochemistry students. To address this issue, a literature search was used to identify potential visualization skills which were used to develop probes in the context of biochemistry. These probes were administered to final year biochemistry students. Analysis of the results utilized the Rasch model to generate an item difficulty map. The results showed that relevant visualization skills can be identified and measured in students, and then ranked according to level of difficulty. The results also revealed that visual literacy requires context-specific propositional knowledge, and is multifaceted in nature in that it is composed of several visualization skills.