The liver is a major organ responsible for performing xenobiotic metabolism. In this process, xenobiotic is uptaken and processed in hepatocytes and subsequently excreted into the bile canaliculi. However, the intracellular heterogeneity in such metabolic processes is not known. We use the molecular probe 6-carboxyfluorescein diacetate (6-CFDA) to investigate xenobiotic metabolism in hepatocytes with intravital multiphoton fluorescence microscopy. 6-CFDA is processed by intracellular esterase to fluorescent 6-CF, which can be imaged and quantified. We found that compared to the nucleus, cytoplasmic 6-CF fluorescence intensity reached a maximum earlier (cytoplasm: 11.3 ± 4.4 min; nucleus: 14.7 ± 4.9 min) following 6-CFDA injection. We also found a slight difference in the rate of 6-CFDA metabolism as the rates of 6-CF decay at rates of 1.43 ± 0.75 and 1.27 ± 0.72 photons/min for the cytoplasm and nucleus, respectively. These results indicate that molecular transport to the nucleus is additionally hindered and can affect drug transport there