The work describes the assay of the potency of insulin (drug substance) and its formulations by gradient elution reversed-phase high-performance liquid chromatography (HPLC). The methods are shown to differentiate between insulins of bovine, porcine and human sequence, and to be both reproducible and stability-indicating. The HPLC assay results show good agreement with those obtained by the mouse blood glucose assay. The advantages of the HPLC assays over the animal response assays are discussed. It is suggested that the animal response assays should now be replaced by the HPLC assays.