Cholesterol is an animal sterol required for membrane structure, and formation of biologically important compounds such as steroid hormones, vitamin D, bile acids. Serum cholesterol estimation is an integral part of lipid profile, and the levels increase with advancing age. Zak’s method employing ferric chloride-acetic and sulphuric acid reagents is explained in this chapter. Nowadays clinical laboratories use cholesterol oxidase-peroxidase enzymatic method. Serum cholesterol level is increased in diabetes mellitus, hypothyroidism, obstructive jaundice, nephrotic syndrome and primary hyperlipoproteinemias, while it is decreased in hyperthyroidism, anemia, malabsorption, and hemolytic jaundice.