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Optic neuritis is an inflammatory optic neuropathy that affects many patients with multiple sclerosis (MS) at some point during their disease course. Differentiation of acute episodes of MS-associated optic neuritis from other autoimmune and inflammatory optic neuropathies is vital for treatment choice and further patient management, but is not always straightforward. Over the past decade, a number of new imaging, laboratory and electrophysiological techniques have entered the clinical arena. To date, however, no consensus guidelines have been devised to specify how and when these techniques can be most rationally applied for the diagnostic work-up of patients with acute optic neuritis. In this article, we review the literature and attempt to formulate a consensus for the investigation of patients with acute optic neuritis, both in standard care and in research with relevance to clinical treatment trials.