A spectrophotometric and thermodynamic method is described for the assay of glibenclamide. The method is based on charge transfer complexation reaction of glibenclamide as n-electron donor with 2,3-Dichloro-5,6-Dicyano-1,4-benzoquinone (DDQ) as π-acceptor to form highly coloured charge transfer complex with a stoichiometric ratio of 1:1. The coloured product was analyzed at 474 nm under the optimized experimental conditions. Beer’s law was obeyed over the concentration range of 20-100 μg/ml. The apparent molar absorptivity was calculated to be 0.99 × 103 L.mol-1.cm-1. Corresponding Sandell’s sensitivity, detection and quantification limits were also reported. Also, the association constant, free Gibb’s energy, enthalpy and entropy changes of the complex were determined. The proposed method was applied successfully to the determination of glibenclamide in pure and commercial forms with good average recovery of 98.24%. Statistical comparison was done using student t-test and f-test at 95% confidence level.