Analysis of biliary metabolites is essential to predict pharmacokinetics and hepatotoxicity during drug development. In this paper, we present a hepatocyte culture configuration that enables the direct recovery of bile acid that accumulates in bile canaliculi by embedding the hepatocytes in a 3D micropatterned collagen gel substrate. We investigated the formation of bile canaliculi in hepatocytes embedded in circular microcavities of various sizes and made from collagen gel. Image analyses using fluorescently labeled bile acid revealed that the area of bile canaliculi in embedded hepatocytes in a microcavity of 60 or 80 μm in diameter was enlarged when compared with other sized microcavities and those of hepatocytes cultured using conventional hepatocyte sandwich cultures. We successfully recovered bile acid from the enlarged bile canaliculi of hepatocytes cultured in microcavities using a glass capillary and quantified the amount recovered. Using our approach, the direct recovery of biliary metabolites, using hepatocyte cultures with enhanced biliary excretion and geometrically enlarged bile canaliculi, may enable accurate screening of pharmacokinetics and drug-drug interactions against drug transporters.