The reaction of tobramycin with o-phthaldialdehyde (OPA) in the presence of 2-mercaptoethanol was studied kinetically by using stopped-flow mixing methodology. New data on the kinetics of this reaction were exploited to develop a simple, fast method for tobramycin determination. The linear range (0.05–30.0 μg mL−1), detection limit (0.03 μg mL−1), precision (&RSD 1.92) and selectivity of the proposed kinetic method are quite acceptable. The method was satisfactorily applied to the analysis of pharmaceutical preparations with no pretreatment.