The electrochemical behavior of anti-hyperglycemic (glibenclamide, GBC) was investigated and discussed at HMDE in Britton-Robinson universal buffer using cyclic voltammetry. Its voltammogram at pH values 6-11 displayed a quasi-reversible redox couple one-electron peak corresponding to a catalytic hydrogen process. Based on this voltammetric peak, a validated and sensitive SW-AdCS voltammetric method was depicted for determination of GBC in the bulk form, diabetic drug (Daonil 5 mg) and in human serum. The (LOD) of 6×10-9 M (2.964 ng mL1) and (LOQ) of 2×108 M (9.88 ng mL1) bulk GBC were achieved, respectively, While for protein-free GBC-spiked human serum were (LOD) of 1.5x10-8 M (7.41 ng mL-1) and limit of quantification (LOQ) of 5x10-8 M (24.7 ng mL-1), respectively. The described analytical method was utilized to estimate the pharmacokinetic parameters of GBC in plasma of hospitalized volunteers after the administration of a single oral dose of Daonil 5 mg (p  0.05).