In this work, intravital multiphoton microscopy is applied to the imaging of liver metabolism with the least invasiveness. We observed intravital dynamics of the uptake, processing and excretion of the organic anion species, 6-carboxyfluorescein diacetate (6-CFDA) in the hepatobiliary system. This is achieved by the use of multiphoton microscopy and an in vivo hepatic imaging chamber which allows us to image the dynamics of hepatic metabolism. Multiphoton images revealed that the hepatic processing of 6-CFDA is completed within approximately 50 minutes. The images reveal the liver metabolism of the uptake and processing of 6-CFDA from the hepatocytes, and the subsequent excretion into bile canaliculi. Our results suggest that this approach is a promising technique for investigating intravital hepatic physiology, diseases, and metabolism.