Abstract: OBJECTIVE To establish a method for the simultaneous determination of 4 flavonoids in Psidium guajava leaves. METHODS HPLC was performed on the column of Eclipse XDB-C₁₈ with mobile phase of methanol-0.2% phosphate(gradient elution) at a flow rate of 1.0 mL·min^-1, the detection wavelength was 360 nm, column temperature was 35℃ and injection volume was 10 μL. RESULTS The linear range was 0.155-3.100 μg for hyperin(r=1.000 0), 0.159-3.182 μg for quercetin-3-O-β-D-arabinopyranoside (r=0.999 8), 0.180-3.601 μg for quercetin-3-O-α-L-furanarabino side ethyl acetate (r=0.999 8),0.007-0.135 μg for quercetin (r=1.000 0); RSDs of precision, stability and reproducibility were lower than 2.0%; recoveries were 100.32%(RSD=1.44%, n=6), 97.79%(RSD=1.11%, n=6), 100.73%(RSD=1.43%, n=6), 98.33%(RSD=1.48%, n=6), respectively. There were quite large differences in the contents of 4 flavonoids in Psidium guajava leaves from different habitats. CONCLUSION The method is simple with good specific, precision and repeatability, can be used for the quality control of Psidium guajava leaves from different habitats.