Background & Aim: Conventional cytological evaluation (CCE) fails to identify nature indeterminate biliary duct stricture (IBDS) in many cases. Digital image analysis (DIA) has the ability to identify and analyze the DNA content of cells. This study assesses the role of DIA in recognizing the nature of IBDS compared to CCE. Methods: A prospective observational study was conducted at the Al-Rajhi University Hospital. Fifty patients with IBDS, based on abdominal imaging, were subjected to endoscopic retrograde cholangiopancreatography (ERCP) and brush sampling. These samples were evaluated with CCE and DIA. Follow-up for at least 9 months and costanalysis had also been done. Results: Based on the final diagnosis, 32 (64.0%) patients had malignant stricture, and 39 (78.0%) had distal stricture. DIA had 84.40% (95% CI; 67.20-94.70) sensitivity and 94.40% (95% CI; 72.70-99.90) specificity in identifying nature of IBDS, whereas CCE had 19.0% (95% CI; 7.20-36.40) sensitivity and 89.0% (95% CI; 65.30-98.60) specificity. Combination of both modalities had 84.40% (95% CI; 67.20-94.70) sensitivity and 83.30% (95% CI; 58.60-96.40) specificity in identification nature of IBDS. Based on CCE alone, only 6/32 ( 18.80%) of malignant stricture were diagnosed, and 26/32 (81.20%) were missed. However, DIA alone was able to diagnose 27/32 (84.40%) of malignant stricture, and only 5 cases were missed. Both procedures had detection rate of malignant stricture as DIA alone. Benign stricture was correctly diagnosed in 16/18 (88.80%), 17/ 18 (94.40%), and 15/18 (83.30%) using CCE alone, DIA alone, and both procedures together, respectively. Cost per detection additional one malignant stricture using DIA required 99.4$. Conclusion: DIA is substantially better than CCE in diagnosing the nature of IBDS but at an increase cost and thus suggests its application in a wider role in clinical practice. Clinical trial number: NCT04112030 (