Multiple sclerosis (MS) is a neurodegenerative disease that affects the central nervous system which produces abnormalities in visual function, as disturbed pupillary responses, even after an episode of optic neuritis (ON). The aim was to assess different parameters of the pupillary response in MS subjects with and without ON. Therefore, 24 eyes of healthy age-matched subjects were included, 22 eyes of subjects with MS (MS group), and 13 subjects with MS with previous ON (MSON group). Pupillary parameters (ratio pupil max/min; latency; velocity and duration; contraction and dilation; and amplitude of contraction) were recorded with the MYAH topographer. Statistical analysis was performed by IBM SPSS Statistics, and parametrical or non-parametrical tests were used according to the normality of the data. MS patients did not significantly differ from healthy patients in any of the parameters analyzed (p > 0.05). Only patients with previous ON were different from healthy patients in the amplitude (40.71 ± 6.73% vs. 45.22 ± 3.29%, respectively) and latency of contraction (0.35 ± 0.13 s vs. 0.26 ± 0.05 s, respectively). The time to recover 75% of the initial diameter was abnormal in 9% of the MS subjects and 12% of MSON subjects. Based on the results of this study, the contraction process, especially latency and amplitude, was found to be affected in subjects with MS and previous ON. The degree of disability and the relation of the decrease in pupil response with other indicators of MS disease should be further investigated considering other comorbidities such as ON in the affection.