Abstract: An ion pair reversed phase High-Performance Liquid Chromatography (HPLC) method has been developed for the simultaneous determination of benzalkonium chloride (BAC) homologues, dodecyl dimethyl benzyl ammonium chloride (C₁₂), tetradecyl dimethyl benzyl ammonium chloride (C₁₄) and cetyl dimethyl benzyl ammonium chloride (C₁₆), in complex chemical disinfectant. Separation was carried out on Platisil ODS column (5 μm, 250 mm×4.6 mm), using methanol and 200 mmol/L hexane sulfonate sodium aqueous solution consisting of 1% triethylamine (V/V), adjusted with perchloric acid to pH 6.0 (85∶15, V/V) as the mobile phase in an isocratic elution. The detection wavelength was 220 nm. In the experiment, sodium hexanesulfonate was used as the ion-pair reagent to enhance the retention of benzalkonium chloride, and triethylamine was used as the tailing-suppressor to restrain the adsorption of benzalkonium chloride to the residual silanols on the stationary phase surface. Calibration curves for C₁₂, C₁₄ and C₁₆ were linear in the range of 0.002-5 mg/mL, 0.005-10 mg/mL and 0.005-10 mg/mL, with the limits of quantifications (LOQs) being 0.0005, 0.001 and 0.001 mg/mL, respectively. The method presents excellent accuracy and satisfactory precision, and it has a significant advantage in the detection of low content samples. The average recoveries were in the range of 99.3%-104.1%, and the intra- and inter-day relative standard deviations (RSDs) were not more than 2.0% for the determined compounds in benzalkonium chloride disinfectant and benzalkonium chloride towelette.