A simple and sensitive stability indicating HPTLC method has been developed and validated for estimation of Palonosetron hydrochloride. Separation of the drug was carried on aluminium plates precoated with silica gel 60 F254 using Ethyl acetate: Methanol: Triethylamine (6:3:1 v/v/v) as mobile phase. The retention factor (Rf) for Palonosetron hydrochloride was found to be 0.50 ± 0.04. The detection was carried at 242 nm. Stress testing of Palonosetron hydrochloride was carried out according to the International conference on harmonization (ICH) guideline Q1A (R2). The drug was subjected to acid, base, neutral hydrolysis, oxidation, thermal degradation and photolysis. Palonosetron hydrochloride showed considerable degradation under oxidative condition. The method was successfully validated according to ICH guidelines Q2 (R1). The data of linear regression analysis indicated a good linear relationship over the range of 250–1500ng/band concentrations with correlation coefficient 0.994. The accuracy of the method was established based on the recovery studies. The LOD and LOQ were 11.81ng/band and 35.78ng/band respectively.