Understanding lipid metabolism and its regulation requires information on the rates at which lipids are produced within the body, absorbed (dietary lipids) into the body, transported within the body, and utilized by various tissues. This article focuses on the use of stable isotope-labeled tracers for the quantitative evaluation of major pathways of fatty acid and triglyceride metabolism in humans in vivo. Adipose tissue lipolysis and free fatty acid appearance in plasma, fatty acid tissue uptake and oxidation, and hepatic very low-density lipoprotein triglyceride secretion are among the metabolic pathways that can be studied by using stable isotope labeled tracers, and will be discussed in detail. The methodology has been in use for many years and is constantly being refined. A variety of tracers and analytical approaches are available and can be used; knowing the advantages, assumptions, and limitations of each is essential for the planning of studies and the interpretation of data, which can provide unique insights into human lipid metabolism.