Abstract: OBJECTIVE To establish an HPLC method for the determination of 5-hydroxymethylfurfural(5-HMF) in ribavirin and glucose injection. METHODS The HPLC method was performed on a Diamonsil C18 column(250 mm×4.6 mm, 5 μm) with 284 nm as the detection wavelength, and methanol-water(30∶70) was used as mobile phase. The column temperature was room temperature, and the flow rate was 1.0 mL·min^-1. RESULTS Ribavirin and excipients did not interfere with the determination of 5-HMF. The regression results which was based on weighted least square method showed a good linearity within the range of 0.099 4-7.949 µg·mL^-1 and 0.002 0-0.198 7 µg·mL^-1 (r=0.999 0 and 0.999 8, respectively) for 5-HMF. The LOQ and LOD of 5-HMF were 0.04 and 0.012 ng, respectively, and the average recovery was 100.4% with RSD of 1.8% (n=6).CONCLUSION The HPLC method was accurate, sensitive and suitable for determining the content of 5-HMF in ribavirin and glucose injection.