High performance liquid chromatography-ultraviolet (HPLC-UV) detection method was performed to simultaneously determine three preservatives: cresol, chlorocresol and benzethonium. According to the current Korean Pharmacopeia Codex, the only-available analytical method for discerning the three preservatives uses thinlayer chromatography, which does not provide accurate detection or quantification. Therefore, this study focused on developing a new HPLC-UV method which gives fast, accurate and sensitive outcomes. The experiment was done using Phenomenex Gemini column [C18 ($250{ imes}4.6mm$, $5{mu}m$)] and the mobile system was methanol and 0.01 M potassium phosphate (monobasic) in water (pH = 3.3). The developed method was validated through linearity, range, system suitability, precision, accuracy, recovery and inter-laboratory variation.