It is essential to teach students about experimental design, as this facilitates their deeper understanding of how most biological knowledge was generated and gives them tools to perform their own investigations. Despite the importance of this area, surprisingly little is known about what students actually learn from designing biological experiments. In this paper, we describe a rubric for experimental design (RED) that can be used to measure knowledge of and diagnose difficulties with experimental design. The development and validation of the RED was informed by a literature review and empirical analysis of undergraduate biology students' responses to three published assessments. Five areas of difficulty with experimental design were identified: the variable properties of an experimental subject; the manipulated variables; measurement of outcomes; accounting for variability; and the scope of inference appropriate for experimental findings. Our findings revealed that some difficulties, documented some 50 yr ago, still exist among our undergraduate students, while others remain poorly investigated. The RED shows great promise for diagnosing students' experimental design knowledge in lecture settings, laboratory courses, research internships, and course-based undergraduate research experiences. It also shows potential for guiding the development and selection of assessment and instructional activities that foster experimental design.