OBJECTIVE The determination method of histatins 5 in human saliva with reversed-phase high performance liquid chromatography (HPLC) was developed. METHODS Salivary samples were collected and diluted with phosphate buffer (pH 2.5). The upper solution was determined with HPLC. Phosphate buffer (pH 3.5) of the mobile phase and C18 column was used throughout the experiment. The detection wavelength was 276 nm. RESULTS The linear ranges were 1.0-50.0 microg x mL(-1). The detection limit was 0.12 microg x mL(-1). The relative standard derivations (RSD) of standard solution for reserved time and peak area were 0.68% and 4.13% respectively. The proposed method was applied for analysis of salivary samples and the satisfactory results were obtained. RSD for sample determination was 4.41% and the average recoveries were 88.4%-109.0%. CONCLUSIONS The method was quick, simple and accurate. Analytical time was less than 15 min. It was adapted for analysis of salivary histatins 5 in salivary samples.