The diagnosis of Rotor syndrome is usually made during childhood or adolescence, based on the presence of skin and scleral jaundice with no pruritus, unlike other cholestatic syndromes. But the disease can also be present in newborns, where high levels of bilirubin can lead to encephalopathy and kernicterus if bilirubin level is above 20 mg/dL. The latter can also be encountered in young adults with increased unconjugated bilirubin, as a consequence of Rotor syndrome or an enzyme such as bilirubin-UDP-glucuronyltransferase deficiency. Icterus may be permanent or intermittent. The disease may have acute episodes, characterized by abdominal pain and low-grade fever. Between acute episodes, the clinical examination of Rotor syndrome patients is normal, other than the presence of icterus.
Because of the clinical similarities, Rotor syndrome and Dubin-Johnson syndrome were thought to be the same pathological entity, until paraclinical data in the late 1970s proved that they were, in fact, different conditions .
Rotor syndrome remains at this time a diagnosis of exclusion.
Blood workup usually shows no abnormalities of aminotransferase, gamma-glutamyl transpeptidase, prothrombin time and alkaline phosphatase levels, although occasional mild increases may be found. Blood bilirubin level is high, usually 2 to 5 mg/dL, but may be as high as 20 mg/dL. Serum bile acid levels are normal. Hemolysis is absent in typical cases, although cases with concomitant hemolytic disorders, such as G-6-PD deficiency and beta thalassemia were described. Still, it can be useful to perform hemolysis tests, such as hemoglobin, reticulocyte count, haptoglobin level and blood smear, which will all be normal. Plasma clearance of bromsulfthalein is delayed because the capacity to transport this product into the bile is reduced by half .
Urinary workup shows markedly increased concentrations of total coproporphyrins and coproporphyrin I. Urinary coproporphyrin is around 65% in homozygotes and 40% in heterozygotes. Coproporphyrin I will account for 65% of total coproporphyrin level, the rest consisting of coproporphyrin III.
Imaging methods used in Rotor syndrome consist of ultrasonography and cholecystography. Hepatic ultrasound imaging is normal in this condition . It should be performed in order to exclude extrahepatic biliary obstruction. Cholecystography is able to visualize the gall bladder in Rotor syndrome. In doubtful cases, a cholescintigraphy with 99mTc-dimethyl acetanilide iminodiacetic acid or Technetium-99m-mebrofenin scintigraphy  can be performed. These investigations will show a faint uptake, similar to patients with hepatocellular disease. Differentiation between these two categories will be done using blood workup.
Less used tests in this condition include liver biopsy and genetic panel. If a liver biopsy is considered to be necessary, normal liver histology will be found in Rotor syndrome. No hepatocyte pigment deposits are described in this condition. Liver biopsy is also useful for excluding other conditions. Genetic tests in affected individuals can detect two mutations in the SLCO1B1 gene and SLCO1B3 gene. Simultaneous mutation in both genes is necessary in order to produce the disease . Genetic differential diagnosis with Dubin-Johnson syndrome is also possible  .