To characterize the activation of pancreatic zymogens (trypsinogen, chymotrypsinogen, and proelastase 1) in acute pancreatitis, we studied the activation of pancreatic juice with porcine enteropeptidase in vitro, and then enzymatic activities and the generation of pancreatic stone protein (PSP) S1form in pancreatic juice were investigated. Further, we determined immunoreactive trypsin, immunoreactive elastase 1, PSP, and α2-macroglobulin-trypsin complex-like substance (MTLS) levels in plasma to which the activated juice was added. In the present report, we demonstrate that the plasma MTLS level reflected the activation of pancreatic trypsinogen in pancreatic juice. Further, the generation of PSP S1form was found at an early stage of activation. Therefore, the plasma MTLS level and the generation of PSP S1form may offer new diagnostic information on the amounts of activated proteases and subsequently on the severity of acute pancreatitis.