The measurement of plasma or serum glucose with ortho-toluidine reagent [ortho-toluidine (6% v/v) in concentrated glacial acetic acid, stabilized with thiourea (0.15% w/v)] has been automated using Technicon Corporation's AutoAnalyzer equipment. The extreme sensitivity of this reagent permitted a large reagent-to-sample volume ratio (22.5:1) which effectively eliminated assay interference by protein. Removal of protein by precipitation or by in-line dialysis or correction of results for a “sample blank” was, therefore, unnecessary. Optimal speed, precision, and accuracy were defined and assay results compared with results from standard technics. For 202 random plasma specimens, glucose values determined by the automated ortho-toluidine procedure averaged 10 mg. per 100 ml. less than values determined simultaneously by the automated alkaline ferricyanide procedure. Oral glucose tolerance test plasma specimens showed an analogous bias which appeared to be more strongly influenced by absolute glucose concentration or elapsed time since dosage. For 42 random plasma specimens the automated ortho-toluidine values averaged 3% lower, manual ortho-toluidine values averaged 1.5% lower, and automated ferricyanide values averaged 7% higher than glucose values determined enzymatically by hexokinase-glucose-6-phosphate dehydrogenase. The automated ortho-toluidine procedure approaches the ferricyanide procedure in economy, approaches the enzymatic methods in specificity, and exceeds both methods in convenience and reliability because of the minimal number of steps involved in the assay procedure.