Tissue-type Plasminogen Activator (tPA) is a serine protease produced by endothelial cells. They target plasminogen, an inactive precursor of plasmin and convert the plasminogen into active plasmin, which degrades the fibrin component of thrombus to aid in dissolution of blood clots. Hence tPA is commonly used in treatment of acute Ischemic stroke in the form of recombinant tissue plasminogen activator (rtPA). rtPA is produced using recombinant technology from different types of vectors depending on the quantities required for the treatment of different diseases. Alteplase (intravenous rtPA) is the most common therapy drug used to treat ischemic strokes. This review gives an outline of the in vitro synthesis of rtPA and its usage in treatment of Acute Ischemic stroke.