textlessptextgreatertextlessbr/textgreaterMass spectrometry is an original and powerful tool used to address a wide range of questions in food and nutritional dairy research. One area where the technique is particularly useful is the structural characterization and identification of milk biomolecules, especially proteins. Proteins and peptides are major constituents of milk and play a decisive role in determining nutritional and functional properties of dairy products. Mass spectrometry has gained increasing popularity as part of the toolkit of techniques developed to investigate milk proteins at the proteome-wide scale. The application of mass spectrometry to protein analysis has been revolutionized in the past decade by the development of new soft ionization techniques and high-sensitivity and high-reproducibility mass spectrometers. In this article, we propose to explore the application of mass spectrometry, especially electrospray ionization and matrix-assisted laser desorption/ionization, the two main soft ionization techniques that have complementary advantages for protein identification, posttranslational modification, polymorphism, and degradation. Mass spectrometry analysis of milk lipids and trace elements is also described briefly.textless/ptextgreater