Triacylglycerols (TAGs) are the major component of milk fat, accounting for 97%–98% of the total lipids. Their composition is extremely complex due to the many different fatty acids (FAs) that can be esterified in the three positions on the L-glycerol molecule, which leads to the formation of a large number of TAG regiospecific and stereospecific isomers. Owing to its complexity, milk TAG analysis poses a significant challenge for analytical chemists, and it is often necessary to combine different techniques, including a prefractionation step to obtain a complete characterization of TAGs and their molecular structures. This chapter reviews the relevant advancements and the most widely used methods (chromatographic and nonchromatographic) for studying TAGs in milk fat and collects information about the analytical conditions, detection systems or fractionation methods used for this purpose. Current advances in chromatographic techniques along with the availability of high-resolution mass spectrometry (MS) analyzers have made TAG identification much easier, and the use of enzymatic and chemical methods have increased our knowledge of the positional distribution of FAs on the TAG molecule. Given the growing development of powerful tools for lipidomic analysis, information on the multiplatform approaches currently applied for TAG characterization has been also revised and updated. Peer reviewed