ACTIVASE® is the recombinant form of human tissue-type plasminogen activator (r-tPA), used in the management of acute myocardial infarction and pulmonary embolism. ACTIVASE® is also now approved for treating ischemic stroke. It is produced by expressing the complementary DNA (cDNA) for natural human tPA in Chinese hamster ovary (CHO) cells. TNK-tPA is a genetically engineered variant of r-tPA with enhanced efficacy and lower incidence of bleeding compared with activase. It was created by three site-directed mutations (T103N, N117Q and KHRR296-299AAAA) and, is also cloned and expressed in CHO cells. CHO cells biosynthesize endogenous hamster tPA called CHO-PA. The amino acid sequence of CHO-PA is highly homologous (80% identical) to that of r-tPA. All three thrombolytic proteins exist as heterogeneous isoforms, mainly due to proteolysis/hydrolysis and differential glycosylation. In this report, a reversed-phase HPLC method was developed to support manufacturing process development. This method not only has the ability to resolve the three plasminogen activators from each other, but also is capable of identifying and quantifying different isoforms of each molecule.