This paper presents a pilot study evaluating the effectiveness of a new robotic approach to characterize altered ankle stiffness in multiple sclerosis (MS) population during standing and walking. The approach utilizing a dual-axis robotic platform could accurately quantify ankle stiffness of the pilot MS patient, with reliability as high as that observed in unimpaired individuals. Further, investigation of the quantified ankle stiffness together with sensor measurements of ankle muscle activation and ankle torque enabled understanding of the mechanisms contributing to ankle stiffness modulation. For the pilot MS subject, muscle contracture, weakness, and reduced range of motion were identified as potential contributors to the modulation of ankle stiffness during standing and walking. Upon successful validation of a future study on a larger group of MS patients, this robotic approach is expected to help clinicians better understand a patient’s altered ankle mechanics, identify the underlying neuromuscular deficiencies, and prescribe patient-specific rehabilitation exercises.