Abstract: Acotiamide hydrochloride trihydrate, which is the first gastrointestinal prokinetic agent, has been proven effective in treating functional malnutrition. There is little method in analyzing this new medicine. The only reported method is using High Performance Liquid Chromatography (HPLC), with methanol-monopotassium phosphate-sodium octanesulfonate as the mobile phase. However, this method is time wasting and unstable because of the using of an ion paring agent in the mobile phase. In this paper, a new HPLC method is established to quantitatively analyze industrial acotiamide hydrochloride trihydrate based on its amphoteric structure. The separation is accomplished in a C18 (4.6 mm×150 mm, 5 μm) column with methanol and 20 mmol/L ammonium acetate aqueous solution (pH 6.8) (45:55, V/V) as the mobile phase. The detection wavelength is set at UV 280 nm. The retention time of acotiamide hydrochloride trihydrate is 8 min with good peak shape, and the total analysis time is 23 min. Under the optimized chromatographic condition, the calibration curve of acotiamide hydrochloride trihydrate is linear in the range of 0.0006 and 1.0 mg/mL, detection limit is 0.0002 mg/mL, and both the intra-and inter-day relative standard deviations (RSDs) are lower than 1%. This method contains no ion paring agent, benefiting the rapid balance of chromatographic condition and improving the analysis repeatability and maneuverability. The method can be performed for rapid detection of acotiamide hydrochloride trihydrate and used in the quality control of raw medicine.