A high specific monoclonal antibody (mAb) against pepsin was produced. The mAb recognized pepsin in rennets from calf, kid and lamb sources. An inhibition enzyme-linked immnunosorbent assay (ELISA) for pepsin in rennet was developed. ELISA was validated by comparing analytical results with the standard chromatographic method. ELISA showed a good agreement with the chromatographic method with a fixed bias.