Objective: To analyze the detection validity of serum biochemical markers in the suspicion of acute biliary disease. Study Design: Retrospective study. Setting: Department of Biochemistry, Islam Teaching Hospital, Sialkot. Period: January 2023 till June 2023. Methods: In a retrospective analysis of 70 patients, there were 35 patients suffering from acute biliary pancreatitis and 35 patients were suffering from non-biliary pancreatitis in group A and B respectively. The patients were identified based on high serum amylase levels. Age, gender, and history were examined, and patients with persistent abdominal pain and abnormal amylase levels were included. Computed tomography detected gallstones, and patients with chronic liver issues were excluded. Results: Comparative analysis conducted using Chi-square and Mann-Whitney tests in SPSS 15.0. A significant result was defined as a P value under 0.005. The sample collection was done from Islam teaching hospital Sialkot and the study was conducted at biochemistry lab of Islam medical college Sialkot. Conclusion: The study, involving 70 patients, aimed to predict acute biliary pancreatitis using serum biochemical markers. The two groups, one with acute biliary pancreatitis and other non-biliary, showed significant differences in key markers (total bilirubin, ALT, direct bilirubin, ALP, AST and serum amylase). These findings suggest the potential diagnostic relevance of these markers.