A procedure is presented for determination of tolbutamide in plasma. The method consists of a chloroform extraction of weakly acidified plasma, concentration of the extract to dryness, dissolution of the residue in a measured volume of ethanol, treatment of the resulting solution with Darco G-60, and measurement of the absorbance of the resulting charcoal filtrate at 228 m[mu]. Recoveries of tolbutamide from human and dog plasmas were satisfactory and plasma levels determined by this procedure have correlated well with the hypoglycemic response elicited by the drug.