The in-vitro inactivation kinetics of dibekacin by three semi-synthetic penicillins (carbenicillin, ampicillin and ticarcillin) were studied taking into account the influence of certain factors such as time, the concentration of the semi-synthetic penicillin in the sample and the reaction medium used in the inactivation process. For a dibekacin/βlactam concentration ratio of 1/100, the maximum percentage of inactivation of dibekacin was obtained for ticarcillin (99%), followed by carbenicillin (90%) and finally by ampicillin (65%) over a period of 45 h.For carbenicillin and ampicillin a linear relationship was established between the inactivation constant (K₁) and the concentration of these penicillins in the sample. It was also found that under the same experimental conditions the inactivation percentage in human serum was smaller than that observed in buffered solution, pH = 7.4.It was observed that the degree of inactivation of dibekacin and gentamicin was similar under the experimental conditions employed in this study.