Background: Elevation of CXCL13, a key regulator of B-cell recruitment in cerebrospinal fluid (CSF) is implicated in multiple sclerosis (MS). Objective: to evaluate if measurement of CXCL13 using a highly sensitive assay is of value in acute optic neuritis (ON) patients for the prediction of later MS. Method: CXCL13 was measured by Simoa in two independent treatment-naïve ON cohorts, a training cohort (TC, n = 33) originating from a population-based cohort, a validation cohort (VC, n = 30) consecutively collected following principles for population studies. Prospectively, 14/33 TC and 12/30 VC patients progressed to MS (MS-ON) while 19/33 TC and 18/30 VC patients, remained as isolated ON (ION). Results: CXCL13 was detectable in all samples and were higher in ON compared with healthy controls (HC) (p = 0.012). In the TC, CSF levels in MS-ON were higher compared with ION patients and HC (p = 0.0001 and p<0.0001). In the VC, we confirmed the increase of CXCL13 in MS-ON compared to ION (p = 0.0091). Logistic regression analysis revealed an area under receiver operating characteristic curve of 0.83 [95% C.I: 0.73–0.93]. Conclusions: The highly sensitive CXCL13 Simoa assay demonstrated ability to identify ON patients and separate MS-ON from ION, and predictive diagnostic values indicates a promising potential of this assay.