Biliary atresia (BA) is a life threatening liver disorder in infants characterised by the disruption of the biliary system. Various types of BA are associated with the alterations in the intrahepatic biliary network, a complex three dimensional network of conduits lined by biliary epitheilal cells. However, the lack of methodologies for accurately and consistently quantitate the difference in three dimensional branching patterns has impeded the study. We thus designed a computer based algorithm that quantitatively computes the three dimentional structure of the network. We have used the computational algorithm in combination with the systematic screening of small molecule inhibitors and identified that inhibiting cyclin dependent kinase 5 (Cdk5) led to a biliary branching defect. Inhibition of Cdk5 did not alter the total number of biliary epithelial cells; however, decreased the connections of the intrahepatic biliary network. We further identified a downstream kinase cascade regulated by Cdk5, which in turn regulates the activity of Cofilin, an actin severing protein. We showed that chemical manipulations to the kinase cascade changed the activity of Cofilin and hence influenced the actin dynamics in the biliary epithelial cells. Our results provide a new insight into the Cdk5-mediated kinase cascade in actin remodeling and the branching morphogenesis of the intrahepatic biliary network.