The formation of clots, also known as “thrombosis,” restricts the supply of blood that can cause acute myocardial infarction and ischemic stroke. The reduced blood flow can cause death. The therapeutic measures involving the surgical interventions, such as removing or bypass the blockage, developing alternate collateral vessels for blood supply, and deploying the thrombolytic enzymes such as streptokinase, are an attractive choice. The pharmaceutical applications of streptokinase include treating acute myocardial infarction, acute retinal vein occlusion, arterial thrombosis and embolism, and deep vein thrombosis. Streptokinase is also used fo the revacularization and restoration of antegrade or forward blood flow to enahnce the tissue survival and patient health. Being an indirect fibrinolytic agent, streptokinase interacts with plasminogen to form an active complex with proteolytic activity that can turn plasminogen into plasmin. The activated complex can resolve the blood clots that obstruct the coronary arteries. The early usage of streptokinase formulations was reported for restricting thrombolytic potency. However, adverse effects such as immunogenicity comparatively short half-life, tissue damage, and peripheral bleeding were major concerns. Due to immense potential of streptokinase, researchers continuously worked on the enhancing the quality of the formulation using this enzyme. This chapter highlights the mechanism, applications, and challenge associated with streptokinase applications in pharmaceutical sectors.