Abstract: A reversed phase high performance liquid chromatography with precolumn derivatization was developed for the determination of low concentration 5-aminolevulinic acid (ALA). An Agilent SB-C18 column (250 mm ×4.6 mm i.d., 5μm) was used for separation, and the elution was performed with methanol and 50 mM sodium acetate solution (adjustment of pH to 4.0 with acetic acid). The flow rate was 1 mL.min-1, the temperature of column was 30℃, and the detection wavelength was at 254 nm with the detection time of 10 minutes. The dynamic linear range is 1-1000 mg.L-1, and the correlation coefficient is above 0.9999. The average recoveries of ALA in 3 samples of fermentation were 101.7%. The results agreed well with those of spectrophotometry. So the developed method is simple and reliable for the determination of low concentration ALA.