A rapid method for the determination of vitamin B2 in infant formula milk powder by high performance liquid chromatography (HPLC) was developed. Vitamin B2 was extracted by ultrasonic treatment with water, and the protein in the extract was removed by precipitation with zinc acetate and potassium ferrocyanide. The analysis was performed on a C18 column using 0.05 mol/L sodium acetate:methanol (70∶30, V/V) as the mobile phase. The limit of detection of the proposed method was 0.2 mg/kg, and a linear calibration curve was obtained in the range of 0.01 to 1.0 μg/mL. Recoveries of spiked samples were between 80.4% and 87.2%. The results indicated that this method was quick and convenient and could be used to detect vitamin B2 in infant formula milk powder.