An experiment was conducted to compare two methods, referred to as A and B, for measuring amylase, lipase, trypsin, and Chymotrypsin activities in samples of pancreatic juice and to investigate the effect of freezing and thawing on enzyme activities. Three growing pigs were used in this study. A catheter was surgically inserted into the pancreatic duct and a simple T-cannula was placed in the duodenum to allow the return of collected pancreatic juice. Pancreatic juice was collected for a total of 24 h and 27 pooled 8-h samples were used in the analyses. Following collection and pooling, the samples were frozen at −80°C, due to the large number of samples and analyses, and stored for 2 weeks. The samples were thawed and enzyme activities were determined with Method A. The samples were frozen again at −80°C, stored for 2 weeks, and rethawed and enzyme activities were determined with Methods A and B. There was a direct relationship between Method A and Method B for measuring amylase, trypsin, and chymotrypsin activity (R2= 0.95, 0.81, and 0.90, respectively). However, a poor relationship was observed between a titrimetric and a photometric method for determining lipase activity (R2= 0.42). A second freezing, storage, and thawing did not affect (p>0.3) the concentration of protein or amylase activity in pancreatic juice. However, the activities of Chymotrypsin, trypsin, and, especially, lipase were decreased by 29.0% (p<0.11), 40.4% (p<0.01), and 82.9% (p<0.001), respectively. In conclusion, either Method A or Method B can be used to measure amylase, trypsin, and Chymotrypsin activities in pancreatic juice but a titrimetric method should be used to determine lipase activity. Furthermore, if the activities of lipase, trypsin, and Chymotrypsin are to be determined, the sample should only be frozen and thawed once. If amylase activity will be determined, the sample can be repeatedly frozen and thawed.