Trypsin activity is usually measured using synthetic substrates, principally amide and ester derivatives of the amino acids lysine or arginine. The aim of this study was to compare trypsin activity measurements done with two assays using amide substrates and two assays using ester substrates. The activity of purified commercial enzyme extracts (bovine and Atlantic cod, Gadus morhua) were measured as was the activity in animal tissues (mice and Atlantic cod) at 5, 15, 25, and 35°C. The results clearly showed the potential impact of the substrate choice on the results when comparing different taxonomic groups at different temperatures. The sensitivity, the measurement reproducibility and even the thermal sensitivity of the enzyme varies according to the assay used.