Context: Insulin autoimmune syndrome (IAS), spontaneous hyperinsulinemic hypoglycemia due to insulin-binding autoantibodies, may be difficult to distinguish from tumoral or other forms of hyperinsulinemic hypoglycemia including surreptitious insulin administration. No standardized treatment regimen exists. Objectives: To evaluate an analytic approach to IAS and responses to different treatments. Design and Setting: Observational study in the UK Severe Insulin Resistance Service. Patients: 6 patients with hyperinsulinemic hypoglycemia and detectable circulating anti-insulin antibody (IA). Main outcome measures: Glycemia, plasma insulin and C-peptide concentrations by immunoassay or mass spectrometry (MS). Immunoreactive insulin was determined in the context of polyethylene glycol (PEG) precipitation and gel filtration chromatography (GFC). IA quantification using enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), and IA were further characterized using radioligand binding studies. Results: All patients were diagnosed with IAS (5 IgG, 1 IgA) based on high insulin:C-peptide ratio, low insulin recovery after PEG precipitation, and GFC evidence of antibody-bound insulin. Neither ELISA nor RIA result proved diagnostic for every case. MS provided a more robust quantification of insulin in the context of IA. 1 patient was managed conservatively, 4 were treated with diazoxide without sustained benefit, and 4 were treated with immunosuppression with highly variable responses. IA affinity did not appear to influence presentation or prognosis. Conclusions: IAS should be considered in patients with hyperinsulinemic hypoglycemia and a high insulin:C-peptide ratio. Low insulin recovery on PEG precipitation supports the presence of insulin-binding antibodies, with GFC providing definitive confirmation. Immunomodulatory therapy should be customized according to individual needs and clinical response.