To correlate occlusion red near-infrared spectroscopy technology with intravenous and interstitial glucose levels, occlusion red near-infrared spectroscopy and glucose levels were measured in five subjects with diabetes mellitus during a stepped hyperinsulinemic hypoglycemic clamp. Validation was achieved using a standard error grid and linear correlations. During hyperinsulinemic hypoglycemic clamp linear correlation was significant at r = 0.836, and the intercept was 43.3 mg/dL. Of the glucose measurements, 94.3% were assigned to zones A and B of the standard error grid, the zones of the greatest therapeutic relevance. Expected interference from serum triglycerides, catecholamines, and cortisol was not apparent. These results demonstrate the applicability of occlusion red near-infrared spectroscopy for in vivo glucose monitoring. The technology has implications for continuous non-invasive glucose sensing.