Optical coherence tomography (OCT) allows evaluation of inter-eye differences (IEDs) in peri-papillary retinal nerve fiber layer (pRNFL) and macular ganglion cell-inner plexiform layer (GCIPL) thicknesses to identify unilateral optic nerve involvement (UONI), which is included in the 2024 revised McDonald diagnostic criteria for multiple sclerosis (MS). To evaluate the test-retest reliability of pRNFL and GCIPL thicknesses/IEDs in people with MS, other neurological disorders, and healthy controls using Cirrus HD-OCT. 509 participants underwent Cirrus HD-OCT, acquiring two macular and optic disc scans per eye within each session. Scans meeting OSCAR-IB quality control criteria were included in final analyses (959 eyes), with no clinical/demographic exclusions (reflecting a real-world clinical setting). Reliability was assessed using coefficients of variation (COVs), intraclass correlation coefficients (ICCs), and Bland-Altman limits of agreement (LOA). IED consistency was evaluated using difference-in-differences (DiDs) of test-retest measurements. GCIPL demonstrated superior reliability (ICC: 0.998, COV: 0.40%, LOA: -1.29 to 1.35 μm) to pRNFL (ICC: 0.989, COV: 1.18%, LOA: -3.59 to 3.70 μm) thickness. Inter-eye absolute DiDs [pRNFL: 2.00 μm (standard deviation (SD) 1.73); GCIPL: 0.64 μm (SD 0.67)] were lower than IED thresholds proposed for identifying UONI. The excellent reliability of GCIPL and pRNFL thicknesses/IEDs support OCT for identifying UONI to diagnose MS.