Prazosin hydrochloride is a sympatholytic alpha-adrenergic blocker used in the treatment of anxiety, hypertension, refractory pulmonary oedema and panic disorders. Rapid, efficient, cost effective and reproducible isocratic reversed phase method has been developed and validated for the determination of prazosin in active pharmaceutical ingredient, dosage formulation and human serum using mobile phase 75:25 v/v acetonitrile:water having pH 3.20 adjusted with glacial acetic acid. Mobile phase was pumped at a flow rate of 1.5 mL min−1 using gradient elution through prepacked Nuclosil, C18 (250×4.6 mm, 10 μm) column. UV detection was performed at 250 nm. Method was validated following the ICH guidelines. Calibration curve was linear in concentration range 1.0-10 μg mL−1 with correlation coefficient 0.9999, and lower limits of detection and quantitation as 3.3 and 2.2 ng mL−1 and 9.8 and 6.1 ng mL−1 in raw material and serum respectively. Recovery was found to be in the range 99-100% and precision less than 1%. Developed method was successfully applied for routine analysis of drug pharmaceutical formulations and serum and also to study the interaction of prazosin with metal essential to human body at physiological temperature (37°C).