An analytical method was developed for the determination of casein phosphopeptides (CPP) in powdered formulae for infants and young children by high performance liquid chromatography. Three pretreatment methods and two operating conditions were optimized. The results showed that the optimal pretreatment conditions were as follows: transferring samples to a 50 mL volumetric flask after pH adjustment to 4.6 with 1 mol/L hydrochloric acid, and then filtering them through a filter paper. The chromatographic separation was carried out by gradient elution using 0.05% trifluoroacetic acid as mobile phase A and 0.05% acetonitrile as mobile phase B. The spiked recovery was between 94.50% and 95.45%, good linearity was observed with R2 = 0.999 8, and the detection limit was 2.0 mg/100 g. The method had the advantages of simple sample pretreatment as well as good stability and reliability, and the results from this method met the requirements in terms of linearity, recovery, accuracy, precision and detection limit. Thus it could be used to determine the content of CPP in powdered formulae for infants and young children.