The purpose of the present study was to evaluate an enzyme linked immunosorbent assay (ELISA) technique for the determination of insulin in canine plasma samples. The test kit Enzymun‐Test®^{Boehringer Mannheim} for determination of human insulin was used in connection with the analyzer system Enzymum‐Test®^{Boehringer Mannheim} System ES‐33. The assay protocol suggested by the manufacturer was slightly modified by including an additional standard having a concentration within the canine reference range for insulin. Further, the incubation period for the immunoreaction and the enzymeassay was increased to 150 minutes and 90 minutes, respectively. When using the modified assay the concentration of insulin in canine plasma samples could be accurately and precisely determined with the exception, that the assay appeared to be somewhat imprecise (intraassay coefficient of variation being 22%) when lipaemic plasma samples were analysed. A slight overestimation of the actual concentrations (28%) was observed, but the measured concentrations of insulin in plasma samples from normal dogs compared favourable to those reported in literature. Eventually, a model for introduction of a species specific reference is discussed.