Vasopressin, nonapeptide, used as an antidiuretic hormone. Very few method has been reported for analysis of Vasopressin from pharmaceutical dosage form. A simple and rapid high performance liquid chromatography (HPLC) method was developed for the quantitative analysis of arginine vasopressin released from polymeric nanoparticles. Chromatographic analysis was performed on an RP C18 column with a mobile phase consisting of acetonitrile and phosphate buffer (13:87 v/v) at a flow rate of 1.6 ml/min at a wavelength of 220 nm,  with a retention time 4.1 min. The method was shown to be specific and linear in the range of 1-50 IU/ml (r2 = 0.9997). Developed method was validated for various evaluation parameters as per ICH guidelines. The method showed no peak interference in presence of formulation excipients. The limit of detection and quantitation were 0.32 and 1.06 IU/ml, respectively. The method was applied to the quantitative analysis of drug to study in vitro drug release from polymeric nanoparticles.