The main sugar of blood is glucose along with other carbohydrate constituents. Hence, ‘blood glucose’ is commonly referred as ‘blood sugar’. Blood glucose estimation is a common test done in all laboratories because it helps in diagnosis, management of diabetes mellitus and is a common prerequisite for any surgery. This chapter covers the methods of estimation, choice of blood specimen, preservation of blood, estimation of blood sugar by Folin-Wu’s method, glucose oxidase method, reagents, and procedure. Glucose oxidase (GOD) acts on glucose to produce gluconic acid hydrogen peroxide. Hydrogen peroxide is producing nascent oxygen by preoxidase (POD). Nascent oxygen further reacts with a chromogen to produce colored product, which is estimated colorimetrically.