The first liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of p-aminohippuric acid and inulin, both typical biomarkers of kidney function. 5-(Hydroxymethyl)furfural, generated from inulin by acid and heat preparation, was used as an inulin substitute for the quantification. Acetaminophen was used as the internal standard. Solid-phase extraction was carried out with 5% methanol as the washing solution to optimize the retention of the analytes and to avoid obstruction of the orifice plate of the mass spectrometer caused by any unreacted inulin residue remaining from the sample preparation process. Chromatography separation was performed on a Symmetry C(18) column and a mobile phase composed of 2 mM ammonium formate and 0.1% formic acid in water (solvent A) and 2 mM ammonium formate and 0.1% formic acid in acetonitrile (solvent B) (30:70, v/v). Detection was performed with a triple-quadrupole tandem mass spectrometer using positive ion mode electrospray ionization in the multiple reaction monitoring mode. The selected transitions were m/z 195.2 → 120.2, 127.1 → 109.1, and 152.1 → 110.0 for p-aminohippuric acid, inulin [measured as 5-(hydroxymethyl)furfural], and acetaminophen, respectively. The linearity ranged from 10 to 140 μg/mL and from 100 to 1,400 μg/mL for p-aminohippurric acid and inulin (r > 0.99), respectively. The precisions and accuracies were all within 12 and 11% for the lower limit of quantification and quality control samples, respectively. This application was proven to be reliable and accurate and was successfully applied to a renal function study.