Background: Indoxyl sulfate (IS) is a protein-bound typical uremic toxin that accumulates in patients with impaired kidney function. The laboratory methods used to quantify serum concentrations of IS require improvement. We report an optimal, cost-effective alternative to the methods currently used. Methods: Serum samples were extracted and deproteinized using acetonitrile and then further purified using dichloromethane. The samples were then analyzed using high-performance liquid chromatography (HPLC) with a fluorescence detector at a flow rate of 1.0 ml/min. The isocratic mobile phase consisted of acetonitrile-0.2% (V/V) trifluoroacetic acid in phosphate buffered saline, pH 2.5 (8: 92, V/V). Detector settings were lambda ex 280 nm/lambda em 390 nm. Results: Under the indicated conditions, IS was well separated. The retention time was approximately 6.59 +/- 0.11 min. The results of the precision and reproducibility tests were <3%, and the recovery rate was greater than 95%, (98.44 +/- 3.21%). Conclusion: HPLC with a fluorescence detector is a simple, sensitive, and reliable method for quantifying IS concentration in the serum of patients with chronic renal failure.