For the detection of lopinavir (LPV) in both its pure form and blood serum, a straightforward, precise, and regulated spectrophotometric approach is suggested. The drug's reaction with the 2,4-dinitrophenylhydrazine (2,4-DNPHz) reagent is the foundation of the suggested procedure. The influence of the reagent volume and the pH function was explored to determine the ideal circumstances, the best reaction time and the optimum temperature for the formation of the azo dye product and it was in compliance with Beer’s law in the range of (2.5-22.5)µg.ml-1at the wavelength of 482nm, the molar, absorptivity, Sandellʼs sensitivity, detection limit and quantitation limit were 3143.75 L.mol-1 .cm-1, 0.20002 μg.cm-2, 0.933 μg.ml-1 and 2.82 μg.ml-1.This method has been successfully applied for the determination of LPV in its blood serum. The kinetic parameters (order and rate of reaction) for this method were calculated, and it was found that the order of drug absorption process is pseudo- second-order. additionally calculated, the activation energy was equal to (204.6 J/mol).