Abstract A semi-batch affinity ligand ultrafiltration system used for the separation of trypsin from a simulated mixture containing trypsin (MW: 23,800) and chymotrypsin (MW: 27,000) has been studied using regenerated cellulose membrane (MWCO: 30,000) and soybean trypsin inhibitor (STI) as ligand. The concentrations of both trypsin and chymotrypsin are 0.02 mg/ml in the mixture while the ratio between STI and trypsin has been maintained at 50:1. Operating pressure of the ultrafiltration module was in the range of 199.4–395.5 kPa. Concentration of trypsin in the ultrafiltration module went down from its initial value to zero within 160 s whereas chymotrypsin took 300 s to go down to zero. Two deterministic mathematical models have been developed and have been compared with the experimental observations.