Publisher Summary The plasminogen activator/plasminogen activator inhibitor (PA/PAI) system is of considerable fundamental importance in elucidating the biochemical mechanisms of many physiological and patho-physiological processes. Although many techniques are available to assess the PA/PAI system, including ELISA and molecular assays, fibrin overlay zymography provides a highly versatile tool for the detection and semiquantitative investigation of PAs, PAIs, and PA/PAI complexes. The chapter discusses monitoring the PA/PAI system such as enzyme-linked immunosorbent assay, molecular methods, substrate gel electrophoresis, fibrin overlay zymography, and advantages of overlay zymography. The chapter also discusses the usefulness of fibrin overlay zymography for the detection of PAs and PAIs in complex biological systems. Several semiquantitative results such as fibrin zymography of PAs, densitometric analysis of PA activity, fibrin zymography of PA induction in cell culture, reverse fibrin zymography for PAIs, and fibrin zymography of PAs from CABG patients are discussed in this chapter. Unique to the overlay method is the opportunity to incorporate into the indicator gel multiple components including activators, inhibitors, antibodies, zymogens, cofactors, and allosteric modifiers. This enables highly sensitive and specific assays to be developed, and thus affords extensive biochemical investigation of diverse proteolytic components including those of the PA/PAI system. The chapter describes the materials and methods required to perform fibrin overlay zymography such as material used, sample preparation, SDS-PAGE, preparation of fibrin indicator gel, fibrin zymography, and reverse zymography.