Copyright © 2018 Nahm et al. Shigella is an important cause of diarrhea worldwide, with serotypes Shigella flexneri 2a, S. flexneri 3a, and Shigella sonnei demonstrating epidemiological prevalence. Many development efforts are focused on Shigella lipopolysaccharide (LPS)-based vaccines, as O antigen-specific conjugate vaccines are immunogenic and efficacious. Immunization with Shigella vaccines containing LPS can elicit antibodies capable of killing Shigella in a serotype-specific manner. Thus, to facilitate Shigella vaccine development, we have developed a serum bactericidal assay (SBA) specific for three Shigella serotypes that measures killing of target bacteria at multiple serum dilutions and in the presence of exogenous complement. The SBA has a high analytical throughput and uses simple technologies and readily available reagents. The SBA was characterized with human sera with bactericidal antibodies against S. flexneri 2a, S. flexneri 3a, and S. sonnei Purified LPS of a homologous serotype, but not a heterologous serotype, inhibited bacterial killing. Assessment of precision found median intra-assay precision to be 13.3% and median interassay precision to be 19 to 30% for the three serotypes. The SBA is linear, with slight deviations for samples with low (~40) killing indices. The SBA was sensitive enough to allow about 100-fold predilution of serum samples. Repeat assays yielded results with less than 2-fold deviations, indicating the robustness of the assay. Assay results from four different laboratories were highly comparable when normalized with a reference serum. The Shigella SBA, combined with a reference serum, should facilitate the development of Shigella vaccines across the field.IMPORTANCEShigella is an important cause of diarrhea worldwide, and efforts are ongoing to produce a safe and effective Shigella vaccine. Although a clear immune correlate of protection has not been established, antibodies with bactericidal capacity may provide one means of protecting against shigellosis. Thus, it is important to measure the functional capacity of antibodies, as opposed to only binding activity. This article describes a simple, robust, and high-throughput serum bactericidal assay capable of measuring Shigella-specific functional antibodies in vitro We show for the first time that this assay was successfully performed by multiple laboratories and generated highly comparable results, particularly when SBA titers were normalized using a reference standard. The serum bactericidal assay, along with a reference serum, should greatly facilitate Shigella vaccine development.