Low titers of blood circulating anti-NMDAR1 autoantibodies have been reported in a significant subset of the general human population. Currently, immunohistochemical staining and cell-based assays are the standard methods for their detection and semi-quantification. However, detection and quantification of these low titers of blood circulating anti-NMDAR1 autoantibodies are problematic because of high non-specific background. Development of a new method to more accurately quantify these low titers of blood anti-NMDAR1 autoantibodies will facilitate studies on their potential impacts on psychiatric symptoms and cognition. We previously reported a robust production of anti-NMDAR1 autoantibodies against the ligand binding domain of NMDAR1. As a proof of principle, we report the development of a novel simple immunoassay for quantification of cross-species blood anti-NMDAR1 autoantibodies and its validation with immunohistochemistry and cell-based assays in both humans and mice. Specificity of our quantification was also investigated.