ABSTRACT: A simple, rapid and precise reverse phase high-performance liquid chromatographic method has been developed and validated for the estimation of Polysorbate 80 in ice cream. Polysorbate 80 was quantified as oleic acid released by alkaline hydrolysis (saponification) of its ester bonds, followed by hexane extraction and reconstitution in acetonitrile. Chromatography was carried out on a C18 column (4.6 × 150 mm, 5 µm) using a mixture of 0.1% phosphoric acid/formic acid in water and acetonitrile (45:55% v/v) as the mobile phase at a flow rate of 1.0 mL/min, and detection was carried out at 195 nm. The retention time of Polysorbate 80 (as oleic acid) was 3.2 min. The method produces linear responses in the concentration range of 50-150 µg/mL (r = 0.9995). The precision of the method, expressed as %RSD, was below 2.0%, the mean recovery ranged from 98.8% to 100.1%, and the LOD and LOQ were 0.57 µg/mL and 1.74 µg/mL respectively. The method was validated for system suitability, specificity, linearity, precision, accuracy, robustness, LOD and LOQ as per ICH Q2(R1) guidelines and was successfully applied to an ice cream sample, in which Polysorbate 80 was found to be 127 mg/kg, within the prescribed limit of 175 mg/kg.