Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from the gastrointestinal tract. It is best known for its glucose-dependent insulinotropic effects. GLP-1 is secreted in its intact (active) form (7–36NH₂) but is rapidly degraded by the dipeptidyl peptidase 4 (DPP-4) enzyme, converting >90% to the primary metabolite (9–36NH₂) before reaching the targets via the circulation. Although originally thought to be inactive or antagonistic, GLP-1 9–36NH₂ may have independent actions, and it is therefore relevant to be able to measure it. Because reliable assays were not available, we developed a sandwich ELISA recognizing both GLP-1 9–36NH₂ and nonamidated GLP-1 9–37. The ELISA was validated using analytical assay validation guidelines and by comparing it to a subtraction-based method, hitherto employed for estimation of GLP-1 9–36NH₂. Its accuracy was evaluated from measurements of plasma obtained during intravenous infusions (1.5 pmol × kg⁻¹ × min⁻¹) of GLP-1 7–36NH₂ in healthy subjects and patients with type 2 diabetes. Plasma levels of the endogenous GLP-1 metabolite increased during a meal challenge in patients with type 2 diabetes, and treatment with a DPP-4 inhibitor fully blocked its formation. Accurate measurements of the GLP-1 metabolite may contribute to understanding its physiology and role of GLP-1 in diabetes.