BACKGROUND Many diagnostic tests for insulin dysregulation use reference intervals established with an insulin radioimmunoassay (RIA) that is no longer available. A chemiluminescent immunoassay (CLIA) is commonly used for the measurement of serum insulin concentration in clinical practice but requires further validation, especially at clinically relevant reference intervals. OBJECTIVE To evaluate the CLIA for measurement of equine insulin and compare it to the previously validated, but now unavailable RIA. METHODS Equine serum samples (n = 78) from clinical and experimental studies. METHODS In this experimental study, performance of the CLIA was evaluated using standard variables, including comparison with the RIA. Continuous and binary outcomes were analyzed. RESULTS The CLIA showed good intra-assay (coefficient of variation [CV], 1.8-2.4%) and interassay (CV, 3-7.1%) precision. Acceptable recovery on dilution (100 ± 10%) was achieved only at dilutions <1:1. Recovery on addition was acceptable. Comparison of the CLIA and RIA showed strong positive correlation (r = 0.91-0.98), with fixed and proportional bias. At 3 diagnostic cutoffs, sensitivity of CLIA compared with RIA ranged from 67 to 100% and specificity from 96 to 100%. CONCLUSIONS The CLIA is a highly repeatable assay which is suitable for within- and between-horse comparisons. Dilution of high concentration samples should be performed with charcoal-stripped serum (CSS) and at the lowest dilution factor possible. At concentrations commonly used for diagnosis of insulin dysregulation (≤100 μIU/mL), results from the CLIA tend to be lower than from the RIA and should be interpreted accordingly. Further standardization of equine insulin assays is required.