Purpose <p>We had a patient with type 2 diabetes (patient A) who suffered from hyperglycemia and frequent episodes of nocturnal hypoglycemia. He had a high titer of insulin antibodies (&gt; 50.0 U/mL) and a very high serum insulin concentration (19,516 µU/mL). The aims of this study were (1) establishing a non-isotopic method to identify insulin analogs that bind less to the antibodies and validating its clinical usage and (2) elucidating the mechanism of his nocturnal hypoglycemia and its management.</p> Methods <p>The optimal conditions for binding study were determined using seven exogenous insulin preparations (human insulin, lispro, aspart, glulisine, glargine, degludec, and detemir) via polyethylene glycol method. The effect of pH on binding of endogenous insulin to its antibodies was examined via gel filtration chromatography (GFC) on different pH.</p> Results <p>Several times higher concentration of exogenous insulin was most appropriate in binding study. It revealed that all seven insulin preparations significantly bound to the insulin antibodies in patient A. Among the other 53 serum samples containing insulin antibodies, about 70% of them had at least one insulin analog that did not significantly bind to their insulin antibodies. GFC revealed that reducing the pH from 7.4 to 7.2 partially dissociated antibody-bound insulin leading to increased unbound insulin.</p> Conclusion <p>We established and validated a non-isotopic method to identify suitable insulin preparations that bound less to insulin antibodies. Correcting respiratory acidosis during sleep might be effective to prevent nocturnal hypoglycemia in some patients with diabetes having insulin antibodies.</p>