Objective To establish a method for the quantitative detection of milk fat globule membrane proteins in raw milk powder based on characteristic peptides using liquid chromatography-tandem mass spectrometry.Methods Characteristic peptides are selected from the proteolytic products of milk fat globule membrane proteins. The quantification of milk fat globule membrane proteins in raw milk powder is performed using multiple reaction monitoring mode. Isotope-labeled internal standard peptides are synthesized to reduce matrix effects and compensate for losses during sample preparation.Results The method's quantification limits range from 0.234 to 0.811 mg/g. The recovery rates at low, medium, and high spiked levels are 82.5% to 105.6%, with relative standard deviations between 2.2% and 10.3%.Conclusion The method demonstrates high accuracy, sensitivity, and specificity, and can meet the requirements for the quantitative determination of milk fat globule membrane proteins at different levels in raw milk powder.