A method for detection and quantification of Glimepiride in human plasma has been developed using anisocratic elution on reversed phase liquid chromatography with ultraviolet detection at a single wavelength(231 nm) and the addition standard method. Glibenclamide was used as an internal standard. This methodinvolves a liquid-liquid drug extraction (Glimepiride and Glibenclamide) from plasma using dichloromethanediethylether (30:70%v/v). Separation was achieved on a C18 reversed phase column and the mobile phaseconsisted of 45% acetonitrile and 55% water (adjusted to pH 3.3±0.1 with acetic acid glacial). The assayhas been validated over a concentration range of 0.001 to 1.0 μg/mL with addition of Glimepiride 0.25μg/mL. Calibration curve was linear in the described concentration range. The reproducibility, stabilityand recovery of the method were evaluated. Determination of Glimepiride in human plasma by HPLC/UV method was accurate and precise with a quantitation limit of 0.2025 μg/mL. The method wassufficiently sensitive for pharmacokinetic studies of Glimepiride in human plasma.