Levels of glycocholic acid are elevated in the bile of cholangiocarcinoma patients, making it a potential biomarker for the disease. The utility of liquid chromatography mass spectrometry based methods for the quantification of glycocholic acid often suffers from time-consuming extraction steps, carryover, and unavailability of blank bile matrix. To overcome the problems, we developed a dilute-and-shoot flow-injection tandem mass spectrometry (standard addition-based) method for quantification of glycocholic acid in bile. Bile was first diluted, followed by spiking the glycocholic acid standard and internal standard into the diluted bile, which was then directly flow-injected (without chromatographic separation) into the electrospray ionization source. Detection was carried out in negative multiple reaction monitoring mode with m/z transitions set at 464.1?74.0 and 401.2?249.1 for glycocholic acid and internal standard, respectively. A standard addition calibration curve was constructed to determine the amount of glycocholic acid present in human bile. The method has been validated with clinical samples according to FDA guidelines. Linearity was achieved in the spiked concentration range of 12.5 to 200 ng/mL with a correlation coefficient of 0.9857. The method has good accuracy, precision, minimal matrix effect, and is sufficiently sensitive enough to quantitate glycocholic acid even after 800,000-fold dilution. In conclusion, a dilute and shoot flow-injection MS/MS method has been developed and validated for the first time, which could be used in routine analysis of glycocholic acid in human bile in a clinical setting.