The objective of this chapter is to discuss anticoagulants and hemoglobin estimation. Blood coagulates when withdrawn from the vessel and anticoagulants are used to prevent blood from clotting. Commonly used anticoagulants are—calcium chelating agents and heparin. The blood for the tests is collected in specific container or vacuum tubes, which have a colored top. It is essential to use appropriate vacuum tubes with proper ratio of anticoagulant. Hemoglobin is a molecule composed of iron containing pigment called heme and protein globin. One of the important features of anemia is the reduction in the hemoglobin. Colorimetric methods, physical method, chemical method, gasometric method, and automated analyzer are different methods of hemoglobin estimation. At the end of this chapter, self-assessment exercise is also provided, which are very helpful for better preparation.