Objectives: We developed an ultrasensitive enzyme immunoassay (ICT-EIA) for insulin autoantibody (IAA) measurements to better understand the pathophysiology of diabetes. Design and methods: We developed ICT-EIA for IAA and measured IAA in 24 patients with type 1 diabetes, 30 patients with type 2 diabetes. 30 patients with methimazole-treated Graves' disease, 20 patients with Hashimoto's disease, 9 patients with hyperinsulinemia, and 73 healthy control subjects. Results: The conventional ELISA identified 3 patients with type 1 diabetes and 2 patients with type 2 diabetes as IAA positive, whereas 15 patients with type 1 diabetes, 7 patients with type 2 diabetes, and 4 patients with methimazole-treated Graves' disease were identified as IAA positive using ICT-EIA. Conclusions: The ICT-EIA is an ultrasensitive and specific assay for IAA, and its use may provide a better understanding of the role of IAA in diabetes onset and progression. (c) 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.