Chromatography is a physical method which is used to separate the mixture. Ingredients which are separated are distributed between two phases (the stationary and mobile phases). At high performance liquid chromatography (HPLC) device for the mobile phase is used liquid and the stationary phase consists of fillers of very fine grains. Surfactants or surface active agents are bifunctional organic compounds that consist of hydrophilic and hydrophobic groups. They are divided into anionic, cationic, nonionic and ampholytic surfactants. Cationic surfactants have a bactericidal, anti-microbial, anti-corrosion properties and are used in industrial, cosmetic and pharmaceutical products. Tyrosur is a local antibiotic. It comes in the form of gel and powder. It is used in the treatment of minor superficial skin injuries. Cetylpyridinium chloride (CPC) is a cationic quaternary ammonium compound. It is used in some mouthwashes, toothpastes, lozenges and gels. It is an antiseptic that kills bacteria and other microorganisms. HPLC was used for cationic surfactant, CPC, determination in real sample (Tyrosur gel). We previously made calibration of CPC in three points using the following concentrations: 1 × 10-5 , 5 × 10-5 and 8.5 × 10-5 to see where was the peak of CPC. The calibration curve for CPC was made on the basis of data for peak heights for every CPC concentration level. During analysis we used the UV detector (SPD - 10 AV) and Acclaim Surfactant column (5μm , 250 × 4.6 mm). We calculated the proportion of CPC in real sample based on the analysis and the resulting data.