OBJECTIVE To establish a HPLC method for determining the content of L-carnosine and its related substances. METHODS The determination of L-carnosine and the separation of its related substances was performed on a Kromasil NH₂ column (4.6 mm×200 mm, 5 μm).The mobile phase consisted of acetonitrile-methanol-40 mmol·L^-1 dibasic potassium phosphate (44∶56, adjusted pH to 6.3 with Phosphoric acid).The flow rate was 1.0 mL·min^-1. Ultraviolet absorption detector was set at 210 nm and column temperature at 35 ℃. RESULTS The linearity of L-carnosine was in the range of 9.8 to 98 mg·L-1 (r=0.999 8). The average recovery was 100.5% with RSD of 1.0%. The related substances of L-carnosine were completely separated from L-carnosine. The limit of detection (LOD) was 1 ng(S/N=3). CONCLUSION A reproducible and specific method is provided for the determination of L-carnosine and its related substances.