In this study, a high performance liquid chromatography (HPLC) method was developed for the determination of lactoferrin in formulas for special medical purposes intended for infants. Lactoferrin from samples was extracted with phosphate buffer. After being purified on a heparin affinity column, the analyte was separated by reversed-phase HPLC using gradient elution with 0.1% trifluoroacetic acid and acetonitrile mixture as the mobile phase, detected by a UV detector and quantitated by the external standard method. Results showed that good linearity was observed for lactoferrin in the concentration range of 50–500 μg/mL. The detection limit of the method was 5 mg/100 g, and the quantitation limit was 15 mg/100 g. The average recovery was 93.9%. The precision expressed as relative standard deviation (RSD) was 0.12%, and the stability RSD was 0.96%. Proficiency testing confirmed the reliability of the method. This method was convenient, fast, accurate, reliable and reproducible, and could effectively detect lactoferrin in formulas for special medical purposes intended for infants.