Milk fat is one of the most complex lipid matrices, with more than 400 different fatty acids (FA) in its composition and most of them at trace levels. In the past decades, important achievements have been achieved in regard to dairy fat FA analysis due to the remarkable development of modern analytical techniques. This chapter provides an overview of how to approach the analysis of milk and dairy products’ FA composition, including the preparation of derivatives and isolation and fractionation of FA. It also gives information about their quantification by gas chromatography (GC or multidimensional GC × GC) and characterization by mass spectrometry (GC-MS or CACI-MS/MS). Complementary methodologies focused on the resolution of cis-and trans-18:1 isomers and conjugated linoleic acid (CLA), such as silver-ion chromatography (Ag⁺-TLC, Ag⁺-HPLC), silver-ion solid-phase extraction (Ag⁺-SPE) or GC columns that contain polar ionic liquid (IL) stationary phases, are discussed throughout the text. A final section dedicated to spectroscopic techniques (near-infrared spectroscopy, NIRS; Raman spectroscopy, RS; and nuclear magnetic resonance spectrometry, NMR) is also included due to their strong potential for real-time and online FA determination and their growing interest over the past few years.