Until recently, neuromyelitis optica (NMO) was considered to be a clinical variant of multiple sclerosis (MS). The discovery of disease-specific anti-aquaporin-4 antibody has facilitated the differentiation of NMO from MS and led to the recognition of a broader phenotypic spectrum now referred to as neuromyelitis optica spectrum disorder (NMOSD). However, distinguishing NMOSD from MS remains challenging, as a subgroup of NMOSD patients are found to be seronegative. The ability to differentiate between NMOSD and MS is critical because these conditions have distinct treatments and prognoses, and the disease-modifying treatment for MS can actually aggravate NMOSD. This review focuses on clinically relevant guidance for diagnosing and differentiating between NMOSD and MS.