A simple, rapid and selective method was developed for estimation of tiropramide from human plasma. The method involves extracting the tiropramide with n-hexane using diphenhydramine hydrochloride as internal standard. Chromatographic separation was carried out on a reversed phase C18 column using mixture of water and acetonitrile as mobile phase with UV detection set at 230 nm. The retention time of internal standard and tiropramide were 5.6 ± 0.2 and 8.3 ± 0.3 min, respectively. The method was validated and found to be linear in the range of 10–200 ng/ml. The co-efficient of variation for intra-day and inter-day accuracy and precision was less than 12.8%. The mean recovery was found to be 89%. An open, randomized, two-treatment, two period, single dose crossover, bioequivalence study in 12 fasting, healthy, male, volunteers was conducted. After dosing, serial blood samples were collected for the period of 12 h. Various pharmacokinetic parameters including AUC0–t, AUC0–∞, Cmax, Tmax, T1/2, and elimination rate constant (Kel) were determined from plasma concentration of both formulations. Log transformed values were compared by analysis of variance (ANOVA) followed by classical 90% confidence interval for Cmax, AUC0–t and AUC0–∞ and was found to be within the range. These results indicated that the analytical method was linear, precise and accurate. Test and reference formulation were found to be bioequivalent.