This test is based on the couplingreaction of urobilirubin with a stable diazonium salt in buffer. A pink to red color proportional to the urobilirubin concentration is generated.

Urobilinogen is formed in the intestine by the action of bacteria on excreted conjugated (direct) bilirubin. A portion of the urobilinogen is absorbed from the gastrointestinal tract into the bloodstream. It returns to the liver where some is re-excreted in bile (enterohepatic circulation), and the rest (via the general circulation) is excreted into the urine. Urine urobilinogen can be increased as an early indicator of moderate hepatic parenchymal damage. Early toxic injury or hepatitis may also cause increased urine urobilinogen. However, if no bilirubin enters the bile no urobilinogen will be produced; thus, with high grade common bile duct obstruction, both urine and fecal urobilinogen will be decreased, but serum and urine bilirubin are greatly increased. Collection time is important because of diurnal variation in urobilinogen excretion. Alkaline pH of urine increases clearance of urobilinogen and increases reliability of results.