A novel simple, sensitive, and rapid isocratic reverse phase high-performance liquid chromatographic (RP-HPLC) method was developed for the quantitative determination of alosetron HCl, a 5-HT 3 antagonist used in the treatment of severe irritable bowel syndrome (IBS) in females. The optimized chromatographic separation was achieved using a stationary phase of Phenomenex ® kromasil C-18 (250 mm × 4.6 mm; 5 μm particle size) column, and mobile phase of 0.025 M disodium hydrogen orthophosphate buffer, pH adjusted to 3.0 with orthophosphoric acid, and acetonitrile in the ratio of 65:35 ( v/v) with a flow rate of 1 mL min −1. The UV detection was carried out at 217 nm. The developed method provided linear responses within the concentration range 100–2000 ng mL −1, and regression analysis showed a correlation coefficient value ( r 2) of 0.997. The HPLC method was validated as per International Conference on Harmonization (ICH) guidelines with respect to selectivity, precision, linearity, and robustness. Limit of detection (LOD) and limit of quantification (LOQ) were found to be 1 ng mL −1 and 5 ng mL −1, respectively.