Lipolysis is defined as the hydrolytic cleavage of ester bonds in triglycerides (TGs), resulting in the generation of fatty acids (FAs) and glycerol. The two major TG pools in the body of vertebrates comprise intracellular TGs and plasma/nutritional TGs. Accordingly, this leads to the discrimination between intracellular and intravascular/gastrointestinal lipolysis, respectively. This chapter focuses exclusively on intracellular lipolysis, referred to as lipolysis herein. The lipolytic cleavage of TGs occurs in essentially all cells and tissues of the body. In all of them, the resulting FAs are utilized endogenously for energy production or biosynthetic pathways with one exception, white adipose tissue (WAT). WAT releases FAs and glycerol to supply nonadipose tissues at times of nutrient deprivation. The fundamental role of lipolysis in lipid and energy homeostasis requires the accurate measurement of lipase activities and lipolytic rates. The recent discovery of new enzymes and regulators that mediate the hydrolysis of TG has made these measurements more complex. Here, we describe detailed methodology for how to measure lipolysis and specific enzymes’ activities in cells, organs, and their respective extracts.