A simple sensitive method for the determination of Pirenzepine in its dosage form and biological fluids by High-Performance Liquid Chromatography with U.V. detector has been developed. A tablet of 25 mg drug was gound, suspended in 10 ml of water, shaken and then filtered. A known volume of the filtrate is adjusted to appropriate concentration. Twenty μl of this solution was injected. Plasma or urine samples were made alkaline with ammonia before extraction with chloroform which was evaporated and the residue was dissolved in the mobile phase, 20 μl of this solution was injected. The determination limit for quantitation was about 1 ug/ml of pirenzepine. Complete separation of the drug was achieved in about 5.4 min. under the present chromatographic conditions.