Glibenclamide, a second generation sulphonyl urea, is to be more efficacious than first generation medications.A common oral anti-diabetic drug used to treat non-insulin-dependent diabetes mellitus is Glibenclamide. As a result of its 4-6 hour biological half-life, it needs to be administered more than once to maintain plasma concentration. This results in discomfort for the patient and changes in plasma medication concentration, which may result in diminished therapeutic effects or harmful effects. With the aid of the solvent evaporation microencapsulation process, controlled release microspheres and PLGA polymer have been prepared. Effects of preparation conditions, drug: polymer ratio (X1), concentration of poly (vinyl alcohol) (PVA) in aqueous phase (X2), homogenization speed (X3) upon the properties of PLGA microspheres containing Glibenclamide were examined. The most effective formulation was derived utilising response surface methodology and optimised polynomial equations. The microspheres were characterized for particle size distribution (PSD), surface morphology, drug excipient compatibility, drug entrapment, and in vitro drug release. The antidiabetic effect of polymeric microsphere formulation of Glibenclamide demonstrated using Alloxan-induced diabetes mellitus model in rats. Glibenclamide microsphere formulation consistently reduced blood sugar levels up to the ninth day, indicating that drug release from the microsphere formulation was controlled up to the ninth day. Based on the findings, it is possible to conclude that Glibenclamide microsphere formulation has an anti-diabetic effect when administered intramuscularly. According to the results, a single dose of 'Glibenclamide microsphere formulation' normalised blood sugar levels in rats.