To assess the possibility of standardization of a commonly used indirect immunofluorescent antibody (IFA) test for detection of Babesia microti antibody in human sera, the results from four reference laboratories were compared. Patients with babesiosis from southern New England (n = 25) and subjects with no history of babesiosis from southern New England (n = 55) and Iceland (n = 50) were enrolled in the study. Anti-Babesia antibody titers were determined in a blinded fashion by IFA test. The range of test results in the four laboratories was 88%-96% sensitivity, 90%-100% specificity, 69%-100% positive predictive value, and 96%-99% negative predictive value. Interlaboratory and intralaboratory concordance ranged from 84% to 85% and 94% to 100%, respectively. This B. microti IFA procedure is a sensitive, specific, and reproducible method for diagnosing babesiosis and is suitable for use as a standard in laboratories testing human sera for B. microti antibody.