Simple, sensitive and rapid methods of lactic acid (LA) quantification in the presence of other short-chain fatty acids in specimens of biological origin were developed to facilitate investigation of metabolism in body tissues and fluids of farm animals, fermentation processes, and ensiled products by reversed-phase high-performance liquid chromatography (RP-HPLC). LA and other acids in assayed samples were converted to their sodium salts (RCOONa). Derivatization of RCOONa was carried out with of a solution of 2,4’-dibromoacetophenone (DBAP) (2.2–2.4 g DBAP/100 ml of acetone or 20 g DBAP/100 ml of chloroform) and triethylamine. The reaction mixture was vigorously mixed and then reacted for 1 h at 45℃. The separation of derivatized acids was performed on a Nova Pak column (4 μm, 300 x 3.9 mm I.D., Waters) using a binary gradient elution program and photodiode detection at 259 nm. Lactic, acetic and propionic acid peaks were eluted at 11.05±0.10, 18.0±0.1 and 19.6±0.1 min, respectively, while the iso-capronic acid peak (an internal standard) at 23.1±0.1 min. The total run time of the HPLC analysis was 59 min. LA detection at 259 nm assures the excellent selectivity and high sensitivity of the proposed method; the limit of detection (LOD) and the limit of quantification (LOQ) were equal to 0.04 and 0.13 ng/ml, respectively. The use of chloroform as a solvent for DBAP in the derivatization of LA in samples followed by RP-HPLC offers the best sensitivity of LA determination in biological samples. The utility of the method was demonstrated by LA analysis in different materials such as silage, fermented dairy and vegetable products, intestinal digesta, blood and etc. The presented methods based on an inexpensive HPLC column, simple and rapid processing of samples provide accurate and sensitive analytical tools for routine determination of LA, particularly in specimens of biological origin.