Serine proteases and their inhibitors play an important role in an umpteen number of biological processes and hence are extensively studied in the past few decades. Purified proteolytic inhibitor from Sapindus trifoliatus was found to inhibit trypsin, plasmin and pronase of the class of serine proteases. Proteolytic enzymes like chymotrypsin, elastase, subtilisin (serine proteases), papain (Thiol protease), pepsin (acidic protease) and thermolysin (Metallo protease) were not affected by Soap Nut Trypsin Inhibitor (SNTI). Class specificity and group specificity exhibited by SNTI can be a positive outcome for targeted therapy. The enzymes were affected by the inhibitor in a stoichiometric manner and the enzyme substrate complex has no effect on the reaction. SNTI strongly interacted with trypsin and plasmin and showed a residual activity of 20% and 50%, respectively. 20 µg of SNTI is capable of inactivating 100 µg of plasmin. An IC₅₀ value of 19.6 µg/mL of purified SNTI acounted for 50% inhibition of plasmin.