In this paper, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to determine the contents of α-lactalbumin, β-lactoglobulin and lactoferrin in milk and infant formula. The sample was dissolved with ultrapure water, and reacted with dithiothreitol and iodoacetamide to open disulfide bonds and simultaneously protect sulfhydryl groups before being hydrolyzed with trypsin. After completion of the reaction, aqueous acetonitrile was used for making up to a constant volume, and then centrifugation was carried out to take the supernatant for quantification by external standard method. The results showed that the calibration curves for all analytes were linear in the range of 0–100 μg/mL, with correlation coefficient (r) greater than 0.995. The average recoveries of the analytes from spiked raw milk were 93.5%–117.1%, with relative standard deviation (RSD) ranging from 1.4% to 6.7%. In conclusion, the method was simple, rapid and sensitive, and could be used for the determination of α-lactalbumin, β-lactoglobulin and lactoferrin in milk and milk powder samples.