A simple, sensitive, precise and specific reverse phase high performance liquid chromatographic method was developed and validated for the determination of Palonosetron Hydrochloride in bulk and softule dosage forms. It was found that the excipient in the tablet dosage forms does not interfere in the quantification of active drug by proposed method. The HPLC separation was carried out by reverse phase chromatography on ODS-UG-5 column (250×4.6mm) with a mobile phase composed of Buffer (0.025M sodium dihydrogen orthophosphate pH adjusted 6.9 with triethyl amine) : acetonitrile (65:35 %v/v) in isocratic mode at a flow rate of 1.0ml/min. The detection was monitored at 240nm. The calibration curve for Palonosetron Hydrochloride was linear from the concentrations of 0.03μg/ml to 2μg/ml. The interday and intraday precision was found to be within limits. The proposed method has adequate sensitivity, reproducibility and specificity for the determination of Palonosetron Hydrochloride in bulk and its softule dosage forms. LOD and LOQ for Palonosetron Hydrochloride were found to be 20ng/ml and 60ng/ml. Accuracy (recoveries: 98.76-100.2%) and reproducibility were found to satisfactory.