Understanding deep evolutionary time is crucial for biology education and for conceptualizing evolutionary history. Although such knowledge might help citizens contemplate their actions in the context of human existence, understanding deep evolutionary time is a demanding cognitive endeavor for students. The enormous magnitudes of evolutionary time are often visually communicated through phylograms and timelines. Given the importance of understanding evolutionary time in various scientific domains at large, there is a need for tools to gauge students’ knowledge about visually communicated deep evolutionary time. In response, we describe the design and validation of an instrument to measure knowledge about the visual representation of deep evolutionary time. Development, expert panel evaluation, and piloting of an initial 14 questions with 139 respondents resulted in a 10-item multiple-choice questionnaire. Subsequent collection and analysis of 212 responses validated the 10-item Deep Evolutionary Time Visual Instrument (DET-Vis). Identification of a single factor suggests a unidimensional construct that represents knowledge about the visual communication of deep evolutionary time. A Cronbach’s alpha of 0.73 yielded an acceptable internal consistency of the instrument. The items of the instrument discriminate well with discrimination coefficients between 0.25 and 0.53. The instrument is of moderate difficulty with difficulty indices ranging from 0.56 to 0.81. The seven-step methodological design and validation procedure of this study yielded a unidimensional, valid, and reliable ten-item deep evolutionary time visual test instrument. The instrument items probe both procedural and declarative aspects of the construct that could warrant future psychometric exploration. Use of DET-Vis in pedagogical practice could help support the teaching of deep evolutionary time at upper secondary and undergraduate levels.