Eltrombopag, which is used to treat thrombocytopenia, causes bilirubin-like yellowish-brown serum discoloration and interferes with biochemical parameter measurements. However, the extent of these interferences and the methods and reagents involved remain unclear. Although previous studies have examined the effects of eltrombopag on bilirubin measurements, its effect on enzymatic measuring of bilirubin levels, which is widely used in Japan, has been limited. Serum samples were collected from 17 eltrombopag-receiving patients. Eltrombopag was added to drug-free serum samples at concentrations ranging from 2.5 to 40.0 µg/mL. The light absorption spectra of the samples were recorded. The lipemia index and 21 biochemical parameters, including total bilirubin (TB) levels, measured by enzymatic, vanadate, and Doumas methods were analyzed. The addition of eltrombopag resulted in a concentration-dependent yellowish-brown discoloration of the serum samples and an increase in absorbance from 340 to 640 nm. The lipemia index significantly increased with increasing eltrombopag concentrations. Fourteen patients displayed triglyceride values below 2.60 mmol/L, one of whom exceeding the lipemia index threshold and exhibiting a yellowish-brown serum with an absorbance increase from 340 to 640 nm. The TB measurements showed a statistically significant positive bias across all three methods, with varying degrees of interference: enzymatic (280.2 %), vanadate (124.6 %), and Doumas (323.9 %) at 40.0 µg/mL eltrombopag. Moreover, uric acid levels showed a statistically significant, concentration-dependent negative bias. This study provides valuable insights into eltrombopag interference, offering well-defined methodological details to assist clinical laboratories in accurately interpreting the patient results.