An ELISA for the detection of porcine pepsin has been developed. Porcine pepsin was detected using this assay at concentrations of ≥1 μg ml‐1. Other cheese rennetting agents, such as bovine rennet orMucor mieheirennet did not cross‐react in the assay either separately or in mixtures. The ELISA was used for the determination of porcine pepsin at concentrations of 1% (10 μg ml‐1) and over in combination with bovine rennet/M.mieheirennet mixtures. The effects on the assay of samples which had been subjected to a pH range between 5.0 and 7.0 (which may be encountered during the storage of rennet mixtures and in cheese making procedures), were tested. Similarly pre‐incubation of porcine pepsin in the presence of NaCl up to 10%, conditions encountered in certain salty cheese preparations, had no subsequent effect on the sensitivity of the assay when such samples were tested. Milk proteins showed no cross‐reactivity with the anti‐porcine pepsin and increasing concentrations of casein to 75 mg ml‐1caused no significant inhibition