Background and Objective: In neuroimmune diseases, autoantibodies are not only used for diagnosis, but also as markers for assessing disease severity and treatment efficacy, and as variables in predicting the prognosis and in subgroup classification. Accuracy of an assay is a prerequisite for using antibodies in the diagnosis and management. The antibody positive subgroup is important in constellating phenotypes, while the antibody negative subgroup provides an opportunity for discovery of new antibodies. This review is to discuss the validation studies of diagnostic accuracy in antibody assays and reasonable definitions of antibody positive/negative subgroups in neuroimmune diseases.Methods: The literature discussing validation studies of diagnostic accuracy in antibody assays and reasonable definition of antibody positive/negative subgroups in autoimmune diseases was broadly searched and discussed.Key Content and Findings: The designs of a validation study, principles in defining the disease spectrum and biases, interpretation of the testing result and specific considerations for validation studies in neuroimmune diseases (such as phenotypes and inclusion criteria, control group selection, dynamic changes of antibody composition and sampling time, relationship between duration and severity of disease and antibody status, and pre-test factor), and improvement of antibody test techniques, as well as definitions and algorithms in defining antibody positive/negative subgroups in clinical practice and researches, are discussed.Conclusions: The successful implementation of validation studies of autoantibody assays in neuroimmune diseases depends on reasonable design, phenotypic profiles of included patients and potential inclusion bias, test result interpretation and the influence from inclusion criteria, control group selection, patient characteristics on specimen sampling and pre-test factors. Reasonable definitions on antibody positive/ negative groups may be different in clinical practice and researches, and rigorous definitions are conducive to constellation of disease phenotype and the exploration of new antibodies within a disease entity.