Cetrimide (CE) is a quaternary ammonium compound and a cationic surfactant, which can be used as an antiseptic and preservative in various formulations. Antiseptic solutions of Cetrimide are available in combination with Chlorhexidine Gluconate (CHG) for external use. Chlorhexidine is a biguanide with high microbicidal activity and is widely known as a skin disinfectant. The present work displays the development and validation of an RP-HPLC isocratic method for the simultaneous determination of CE and CHG. The method consists of a Hypersil® SAS C1 (4.6 × 250 mm) 5 μm column, with a mobile phase of 85%/15% v/v MeOH-NaH2PO4·H2O, aqueous solution. In addition, 0.2% of triethylamine (Et3N) was added to the buffer for the confrontation of peak tailing, and then the pH was adjusted to 3.0 with ortho-phosphoric acid (H3PO4). The flow rate was set at 1 mL/min, and adequate detection was achieved with a diode array detector (PDA) at 205 nm. The method was successfully validated according to ICH guidelines for specificity, linearity, accuracy, precision and stability for sample and standard solutions. In addition, the robustness of the method was evaluated through statistical and graphical analysis, using a fractional factorial experimental design.