The objectives of this study were (1) to develop a method for the determination of 10 uremic toxins (3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), hippuric acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid, kynurenine, p-cresyl glucuronide, p-cresyl sulfate, phenylacetylglutamine and trimethylamine N-oxide (TMAO)), and 3 precursors (tyrosine, phenylalanine, tryptophan) in serum and (2) to compare two separation methods to determine the free serum fraction: rapid equilibrium dialysis (RED) and ultrafiltration (UF). The method was developed on a liquid chromatography system coupled to a tandem mass spectrometer. Fifty µL of serum sample were precipitated with methanol after addition of internal standard. The two separation methods were compared using serum samples from patients suffering from renal impairment (n = 30). The method has been validated according to the European Medicines Agency (EMA) guidelines. Calibration curves were linear from 1 to 50 ng/mL up to 10,000-50,000 ng/mL according to the compounds. The comparison between the two separation methods produced similar results for all compounds except kynurenine, tryptophan (around 30% more with UF) and indole-3-acetic acid (around 30% more with RED). This study has allowed the development and validation of a sensitive and robust assay for the quantification of free and total concentrations of 10 uremic toxins and 3 precursors in human serum.