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Glycocholic acid (GCA) is an important identified biomarker for various hepatobiliary diseases. However, results obtained from routine clinical measurement significantly varied due to the interference from the extremely similar structure analogues, which cause confusion to clinical diagnosis. In this study, a bracketing calibration-based isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method was developed. The established method exhibited good performance criteria through the adoption of isotope dilution, calibrator bracketing, and gravimetric measurements. The satisfactory precision (0.16%–2.01%) and good recoveries were achieved at five spiked levels (98.0–100.9%). The limit of detection (LOD) and limit of quantification (LOQ) were 0.01 ng/mL and 0.05 ng/mL, respectively. No interference, matrix effect, and carryover were observed. The sample preparation process was easier to operate, without further solvent evaporation and time-saving. The optimized method was successfully applied to quantify GCA with a wide range of concentrations in clinical human serum samples and also compared with clinical routine systems.