A novel approach for on-line monitoring of drug metabolism in continuously perifused, precision-cut liver slices (PCLS) in a microfluidic system has been developed using high-performance liquid chromatography with UV detection (HPLC-UV). In this approach, PCLS are incubated in a microfluidic device made of poly(dimethylsiloxane) (PDMS) by continuous, single-pass perifusion with fresh medium. Two syringe pumps are incorporated into the system to infuse substrates or inhibitors at varying concentrations into the perfusion medium just before the chip entrance. The medium containing the metabolites produced by the PCLS is directed toward an injection loop. Once filled, the content of this injection loop is automatically injected onto an HPLC for analysis. The on-line analysis of metabolites was tested by using the substrate, 7-hydroxycoumarin (7-HC). Rapid switching between substrate and solvent control was possible, and a direct metabolic response of the liver slice to perifusion with substrate was detected. Very stable phase II metabolism over a period of 24 h was observed. The inhibitory effect of phloxine B on the formation of 7-hydroxycoumarin glucuronide (phase II product of 7-HC) was also investigated. Phloxine B was injected into the incubation medium in increasing concentrations varying from 0 to 200 μM. The results showed a concentration-dependent inhibition of 7-HC glucuronide formation and allowed the calculation of an IC50 value (concentration in which 50% of the enzyme is inhibited) of ∼85 μM using one single liver slice. On-line detection was also shown to be advantageous for the detection of unstable metabolites. This was demonstrated by determination of the metabolites of the drug diclofenac. The reactive metabolite, acyl glucuronide, was detected at relatively high concentrations which remained very constant over a period of 4 h. In contrast, only low and decreasing amounts of diclofenac acyl glucuronide could be measured in the conventional well-plate incubation system. The advantages of this novel on-line analysis system for PCLS include the capability to obtain direct information about tissue function, assess the concentration dependence of drug-drug interactions in one single slice, and detect unstable metabolites. The system also enables fast analysis without the need to store samples, thus eliminating the associated freeze-thaw problems, and allows the simultaneous analysis of multiple metabolites.