Diagnosis of equine hyperinsulinemia requires an accurate method for quantification of equine insulin concentrations. The objectives of the current study were to compare 2 commercially available techniques for measurement of equine insulin, the radioimmunoassay (RIA) and chemiluminescent immunoassay (CIA). Recovery was poor for both assays, but worse for the CIA. Serial dilution of a high endogenous insulin sample yielded better linearity for the RIA ( r2 = 0.99, P < 0.001) than the CIA ( r2 = 0.92, P = 0.009). Bland–Altman analysis indicated that the CIA was, on average, 91 pmol/l higher than the RIA, with wide limits of agreement (95% limits of agreement: –508 to 691 pmol/l). These findings suggest that results between the assays should not be considered interchangeable.