A sensitive, selective and simple method using a precipitation of protein with 10% perchloric acid, followed by high-performance liquid chromatography (HPLC) with fluorescence detection was developed for the determination of itopride hydrochloride in human plasma, using levofloxacin as the internal standard (IS). Chromatographic separation was obtained within 7.0min using a reverse phase Hypersil BDS C₁₈ (250mm×4.6mm, 5μm) column and an isocratic mobile phase, constituting of a mixture of 0.1mol/l ammonium acetate–methanol (30:70, v/v) flowing at 1.1ml/min. The excitation and emission wavelengths were set at 304 and 344nm, respectively. The method was validated over the concentration range of 5ng/ml to 1000.0ng/ml. The lower limit of quantitation (LLOQ) was 5ng/ml. The extractive recovery of itopride hydrochloride from the biological matrix was more than 80.77%. The intra-day accuracy of the drug containing serum samples was more than 82.94% with a precision of 2.81–4.37%. The inter-day accuracy was 82.91% or more, with a precision of 6.89–9.54%. The limit we have used (70–143%) is based on the local regulatory authority (SFDA). The developed method was validated and successfully applied to bioequivalence studies of itopride hydrochloride in healthy male volunteers.