Long-term ex situ liver perfusion may rescue injured grafts. Little is known about bile flow during long-term perfusion. We report the development of a bile stimulation protocol and motivate bile flow as a viability marker during long-term ex situ liver perfusion. Porcine and human livers were perfused with blood at close to physiologic conditions. Our perfusion protocol was established during phase 1 with porcine livers (n = 23). Taurocholic acid was applied to stimulate bile flow. The addition of piperacillin-tazobactam (tazobac) and methylprednisolone was modified from daily bolus to controlled continuous application. We adapted the protocol to human livers (n = 12) during phase 2. Taurocholic acid was replaced with medical grade ursodeoxycholic acid. Phase 2: Despite administering taurocholic acid, bile flow declined from 29.3 ± 6.5 to 9.3 ± 1.4 mL/h (P < .001). Shortly after bolus of tazobac/methylprednisolone, bile flow recovered to 39.0 ± 9.7 mL/h with a decrease of solid bile components. This implied bile salt independent bile flow stimulation by tazobac/methylprednisolone. Phase 2: Ursodeoxycholic acid was shown to stimulate bile flow ex situ in human livers. Eight livers were perfused successfully for 1 week with continuous bile flow. The other 4 livers demonstrated progressive cell death, of which only 1 exhibited bile flow. A lack of bile flow stimulation leads to a decline in bile flow and is not necessarily a sign of deterioration in liver function. Proper administration of stimulators can induce constant bile flow during ex situ liver perfusion for up to 1 week. Medical grade ursodeoxycholic acid is a suitable replacement for nonmedical grade taurocholic acid. The presence of bile flow alone is not sufficient to assess liver viability.