We describe the development of a new, freely available, online, programmatic-level assessment tool, Measuring Achievement and Progress in Science in Physiology, or Phys-MAPS (http://cperl.lassp.cornell.edu/bio-maps). Aligned with the conceptual frameworks of Core Principles of Physiology, and Vision and Change Core Concepts, Phys-MAPS can be used to evaluate student learning of core physiology concepts at multiple time points in an undergraduate physiology program, providing a valuable longitudinal tool to gain insight into student thinking and aid in the data-driven reform of physiology curricula. Phys-MAPS questions have a modified multiple true/false design and were developed using an iterative process, including student interviews and physiology expert review to verify scientific accuracy, appropriateness for physiology majors, and clarity. The final version of Phys-MAPS was tested with 2,600 students across 13 universities, has evidence of reliability, and has no significant statement biases. Over 90% of the physiology experts surveyed agreed that each Phys-MAPS statement was scientifically accurate and relevant to a physiology major. When testing each statement for bias, differential item functioning analysis demonstrated only a small effect size (<0.008) of any tested demographic variable. Regarding student performance, Phys-MAPS can also distinguish between lower and upper division students, both across different institutions (average overall scores increase with each level of class standing; two-way ANOVA, P < 0.001) and within each of three sample institutions (each ANOVA, P = 0.001). Furthermore, at the level of individual concepts, only evolution and homeostasis do not demonstrate the typical increase across class standing, suggesting these concepts likely present consistent conceptual challenges for physiology students.