This study presents the development and validation of a novel two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) method for the quantitative determination of Ursodoxycholic Acid (UDCA) in human plasma. The method utilizes a two-phase separation system combining Thermo 5CM and 3CM columns, with electrospray ionization (ESI) operating in negative ionization mode. Internal standardization with Valsartan ensured high accuracy and precision. Calibration was performed across a range of 50–8000 ppb, achieving linearity with R² > 0.99 and a weighted regression model (1/x). Analytical validation followed ICH M10 guidelines, evaluating parameters including linearity, sensitivity, matrix effects, and reproducibility. Validation results demonstrated a relative standard deviation (RSD) below 10% for intra- and inter-day analyses. This method was successfully applied to analyze plasma samples from clinical trial participants, demonstrating its robustness and applicability for pharmacokinetic studies of UDCA. This innovative approach offers enhanced resolution and precision, supporting the reliable quantification of UDCA in complex biological matrices.