New kinetic-spectrophotometric method has been developed to determine diazepam (DZP) in pharmaceutical formulations and human control serum. The method is based on ligand-exchange reaction. The reaction was followed spectrophotometrically by measuring the rate of change of absorbance at 425 nm in ethanolic sodium hydroxide solution. The optimum operating conditions regarding concentration of reagents and temperature were established. The initial-rate method is adopted for constructing the calibration curve, which was found to be linear over the concentration range 0.28-3.70 μg mL-1. The optimized conditions yielded a theoretical detection limit of 0.14 μg mL-1 based on the 3Sb criterion. The interference effects of certain drugs, foreign ions and amino acids upon the reaction rate were studied in order to assess the selectivity of the method. The method described in this work was validated following the analytical performance parameters required by the USP-27/NF-22. Statistical Students t-test and F-test have been used and satisfactory results were obtained.