The basis for understanding neurophysiology is understanding ion movement across cell membranes. Students in introductory courses recognize ion concentration gradients as a driving force for ion movement but struggle to simultaneously account for electrical charge gradients. We developed a 17-multiple-choice item assessment of students' understanding of electrochemical gradients and resistance in neurophysiology, the Electrochemical Gradients Assessment Device (EGAD). We investigated the internal evidence validity of the assessment by analyzing item characteristic curves of score probability and student ability for each question, and a Wright map of student scores and ability. We used linear mixed-effect regression to test student performance and ability. Our assessment discriminated students with average ability (weighted likelihood estimate: -2 to 1.5 [theta]); however, it was not as effective at discriminating students at the highest ability (weighted likelihood estimate: >2 [theta]). We determined the assessment could capture changes in both assessment scores (model r[superscript 2] = 0.51, P < 0.001, n = 444) and ability estimates (model r[superscript 2] = 0.47, P < 0.001, n = 444) after a simulation-based laboratory and course instruction for 222 students. Differential item function analysis determined that each item on the assessment performed equitably for all students, regardless of gender, race/ethnicity, or economic status. Overall, we found that men scored higher (r[superscript 2] = 0.51, P = 0.014, n = 444) and had higher ability scores (P = 0.003) on the EGAD assessment. Caucasian students of both genders were positively correlated with score (r[superscript 2] = 0.51, P < 0.001, n = 444) and ability (r[superscript 2] = 0.47, P < 0.001, n = 444). Based on the evidence gathered through our analyses, the scores obtained from the EGAD can distinguish between levels of content knowledge on neurophysiology principles for students in introductory physiology courses.