This study was performed in order to validate an effective high performance liquid chromatograpy (HPLC) method to determine L-carnitine in biological samples such as plasma, milk and muscle in cows. An L-carnitine derivative for fluorescence absorption was synthesized with 1-aminoanthracene (16 mg/mL in acetone) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC; 160 mg/mL in 0.01 M NaH2PO4 buffer) as a precolumn fluorescent derivative reagent. gamma-Butyrobetaine HCI was used as an internal standard. A reversed-phase column with fluorescence detection at the excitation and emission wavelengths of 248 and 418 nm were used. The mobile phase consisted of 30% acetonitrile with 0.1 M ammonium acetate in water (pH 3.5) adjusted with acetic acid and delivered at a flow rate of 1.5 mL/ min. The L-carnitine concentration in plasma, milk and muscle samples of cows after oral feeding with 24 g L-carnitine/day for 2 months was then determined. All biological samples were deproteinated by barium hydroxide and zinc sulfate heptahydrate before the derivative reaction. Blank cow plasma was dialyzed using cellulose membrane for standard calibration. The calibration curve showed good linearity (r2 > 0.999) over the concentration range of 50 to 5000 ng/mL. The precision and accuracy were also satisfactory with less than 15% intra- and interday coefficiency of variations. The peaks of L-carnitine and internal standard in HPLC chromatography were successfully separated in plasma, milk and muscle samples of cows. The current derivatization method of L-carnitine for fluorescence detection was simple and adequately sensitive and could be applied to determine L-carnitine in biological samples.