Neonatal cholestasis (NC) initiates in the first trimester of a newborn, comprising extra and intrahepatic medical conditions, with a high risk of fatality warranting early diagnosis and treatment to prevent morbimortality. The differential diagnosis of NC is a challenge demanding an accurate diagnosis for disease detection. The current study evaluates NC's clinical symptoms and differential diagnosis using ultrasound, liver biopsy, histopathology, and biochemistry. Infant registry data from Hevi Pediatric Teaching Hospital (January 2016 - January 2022) were obtained and screened for subject selection. The inclusion criteria include infants with direct hyperbilirubinemia within an onset of 15 to 90 days of birth. As indicated, the recruited subjects underwent liver ultrasound, blood biochemistry, and liver biopsy. Seventy-two children presented with the criteria for inclusion in the study. The study found that ultrasound helped diagnose 43.1% of subjects for biliary atresia (BA) compared to 34.7% through histopathology. The histopathology confirmed 13 children (18.1%) having neonatal hepatitis. Test sensitivity of the ultrasound method for BA and neonatal hepatitis (NH) was 60% (40.74, 76.6) and 38.46% (17.71, 64.48), respectively. The study found both ultrasound and liver biopsy to be critical diagnostic methods to differentiate the etiology of NC. Ultrasound has a higher specificity and sensitivity for diagnosing BA than NH. Histopathology and blood biochemistry should be considered, too, for effective diagnosis. In the future, larger sample and multicenter studies should be conducted to develop practically implementable strategies.