Lactic acid bacteria (LAB) are considered weakly lipolytic compared with many other groups of bacteria (e.g., Pseudomonas, Bacillus, and Achromobacter). The esterolytic and lipolytic systems of dairy LAB remain poorly characterized. Esterases from lactic acid bacteria, yeasts, and Pseudomonas organisms may be involved in the development of fruity flavors in foods, and pregastric lipase and esterases are essential for the development of typical flavor in Italian cheese. Microbial lipases and esterases may improve quality or accelerate the maturation of cheeses, cured bacon, and fermented sausages. Lipases are defined as glycerol ester hydrolases (EC 3.1.1.3) that hydrolyze tri-, di-, and monoglycerides present at an oil-water interface. Esterases (EC 3.1.1.6) hydrolyze esters in solution and may also hydrolyze tri- and especially di- and monoglycerides containing short-chain fatty acids. Some probiotic strains of LAB can hydrolyze the triglycerides, releasing most short and medium chain, and essential fatty acids, which are valuable to today's health-conscious consumer. Medium chain fatty acids (C6-C14), in particular, have become accepted treatment for patients with malabsorption symptoms, a variety of metabolic disorders, cholesterol problems, and infant malnutrition. These probiotic bacteria could alleviate lipase deficiency in the digestive tract during digestion (steatorrhea). In this chapter, we describe different methods routinely used in our laboratory to determine the esterolytic and lipolytic activity of LAB. These techniques include the use of alpha- and beta-naphthyl derivatives of fatty acids (chromogenic method), the p-nitrophenyl (pNP) derivative of fatty acids (chromogenic method), and triglycerides (agar-well assay technique and titrimetric test) as substrates.