An effective, duplicable, and quick method to determine the purity and formulating form of Cetrimide (CE) have studied and modified utilizing the Reverse Phase High Performance Liquid Chromatography (RP-HPLC). The method was based on the separation of the CE using a reverse phase HPLC Shimadzu LC–20 A, Japan, and Phenomenex C18 column (15 × 4.6mm, 5.0μm). The process was carried out by using the mobile phase ammonium acetate solution 0.1M (AA), methanol (MeOH), and the methyl cyanide in the ratio of (15:60:25 V/V/V), a flow rate of 1.0mL/min, at pH = 3, with 10μL sample loop and the UV - spectrophotometry detector was set at 280nm. However, for the optimum conditions the mobile phase composition, wavelength, pH, and flow rate were studied. The calibration carve has obtained in the range of 10-1000mg/L and the correlation coefficient is 0.9985. The average recovery of the standard drug was 97.56%. The LOD was 5mg/L and LOQ is 16.5mg/L for CE. The process was used successfully to determine the CE in the pharmaceutical forms with the average recovery of 95.61 and 96.02%.