Objective A sensitive and rapid method was developed for the simultaneous determination of L-carnitine and D-carnitine in infant formula by pre-column derivation high performance liquid chromatography， and the key points were explored.Methods Carnitine in sample was extracted with hydrochloric acid， saponified with potassium hydroxide， purified by acetonitrile and solid phase extraction column （SPE） and derivated. The derivative was separated by the C₁₈ chromatography column， detected by UV detector， and quantified by external standard curve.Results The linear ranges of L-carnitine and D-carnitine were 0.2-10 μg/mL with correlation coefficients of 0.999 9 and 0.999 4. The average recoveries of carnitine were 87.14%-96.96% under the 3 spiked concentrations. The limits of detection （LODs） were 0.6 mg/100 g， and the limit of quantification （LOQ） was 2 mg/100 g. The concentration of potassium hydroxide， purification process with SPE， solvent of derivatization reagent and operation in derivation were the key points in this method.Conclusion This method is simple， accurate and is suitable for simultaneous detection of L-carnitine and D-carnitine in infant formula. And there is no significant difference between this method and enzymatic spectrophotometry.