Acute pancreatitis (AP) is a severe inflammatory condition of the pancreas that can result in organ dysfunction, severe illness, and potentially death. However, the absence of a standardized screening test poses challenges in detecting acute pancreatitis. This study aimed to assess the efficacy of two microRNAs, miR-216a and miR-375, as potential biomarkers for acute pancreatic injury. The study also compared their effectiveness with traditional biomarkers, including hemato-biochemical markers, pancreatic ultrasound examination, and histopathological examination, in a canine pancreatitis model six hours after caerulein infusion. Fourteen healthy mongrel dogs were randomly assigned to two groups: the control group (n=7), which received intravenous saline, and the acute pancreatitis group (n=7), which received intravenous caerulein. Dogs in the caerulein group exhibited clinical symptoms such as anorexia, vomiting, diarrhea, and lethargy. Caerulein infusion significantly elevated erythrogram parameters and serum amylase, lipase, ALT, AST, and ALP activities. Molecular analysis revealed a significant up-regulation of miR-216a and miR-375 in the caerulein-treated group compared to the control group. Ultrasonographic examination of the caerulein-treated group demonstrated an enlarged hypoechoic pancreatic structure and a thickened corrugated duodenal wall. Histopathological investigation confirmed the presence of acute exocrine pancreatitis in the caerulein-treated group. The findings of this study demonstrate the potential of miR-216a and miR-375 as effective biomarkers for detecting acute pancreatitis in mongrel dogs. These microRNAs could offer valuable insights into the early diagnosis of acute pancreatitis, complementing the existing diagnostic methods.