Amylase and lipase are digestive enzymes normally released from the acinar cells of the exocrine pancreas into the duodenum. Following injury to the pancreas, these enzymes are released into the circulation. While amylase is cleared in the urine, lipase is reabsorbed back into the circulation. In cases of acute pancreatitis, serum activity for both enzymes is greatly increased. Serum lipase is now the preferred test due to its improved sensitivity, particularly in alcohol-induced pancreatitis. Its prolonged elevation creates a wider diagnostic window than amylase. In acute pancreatitis, amylase can rise rapidly within three to six hours of the onset of symptoms and may remain elevated for up to five days. Lipase, however, usually peaks at 24 hours with serum concentrations remaining elevated for eight to 14 days. This means it is far more useful than amylase when the clinical presentation or testing has been delayed for more than 24 hours. Current guidelines and recommendations indicate that lipase should be preferred over total and pancreatic amylase for the initial diagnosis of acute pancreatitis and that the assessment should not be repeated over time to monitor disease prognosis. Repeat testing should be considered only when the patient has signs and symptoms of persisting pancreatic or peripancreatic inflammation, blockage of the pancreatic duct, or development of a pseudocyst. Testing both amylase and lipase is generally discouraged because it increases costs while only marginally improving diagnostic efficiency compared to either marker alone.