Protease inhibitors (PIs) of Cajanus cajan (L) Millsp. are known for its inhibitory action against trypsin, chymotrypsin and Helicoverpa armigera gut proteinase. These inhibitors were extracted in 1% polyvinyl pyrrolidone, separated by preparative polyacrylamide gel electrophoresis and visualized using gel-X-ray film contract print method. Individual PIs were excised from gel and precipitated in acetone. The purity and inhibitor potency against trypsin were confirmed by sodium dedocyl sulphate-polyacrylamide gel electrophoresis and trypsin inhibitory assay using synthetic substrate Nα-Benzoyl-DL-Arginine-p-Nitroanilide. 100 ug of protein was sufficient for visualization of all inhibitor (PI-1 to PI-9) in 10% polyacrylamide gel solution with high resolution. PI-6, PI-7 and PI-8 showed 22.22 %, 21.77% and 37.77 % inhibition potential against trypsin. The solubility properties of PIs may be altered due to acetone precipitation method. Therefore, use alternate method for purification of PI-1 to PI-5 and PI-9 is needed for further characterization of Cajanus cajan PIs.