Background: Multiple sclerosis (MS) is a demyelinating disease with multiple symptoms. To examine the effect of medications, magnetic resonance imaging (MRI) imaging can be performed. Objectives: This study aimed to compare the visual assessment of MRI images by physicians and the results of a MATLAB-based software developed for evaluating the MS plaque volume. Methods: TYSABRI (natalizumab)-treated patients were enrolled in this study. MRI was performed before and after treatment. A MATLAB-based software was developed to evaluate the MRI images. The images were then subjectively evaluated by three specialists and objectively assessed by the software, and the results were then compared. Results: Thirty-five patients participated in this study. According to the software, the status of 37.14% of the patients improved; however, 62.86% of cases deteriorated after a year of treatment. There was less than 50% agreement between the physicians and less than 55% agreement between the physicians and the software results. A disagreement of almost 45% was observed between the physicians and the software results, as in some cases, small changes were detected in successive images. The second cause of disagreement could be the physicians’ expectations; although the patient’s recovery was highly expected in some cases, their condition deteriorated. Conclusions: The results revealed that the objective assessment of sequential MRI images of MS patients reduced human errors and improved the evaluation of this disease.