This paper details the development of a high-throughput analytical method for the quantification of Ursodoxycholic Acid (UDCA) in plasma samples using a two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) system. The innovative approach integrates an auto-sampler with precise timing to direct analyte flows through two chromatographic columns for enhanced separation. Electrospray ionization in negative mode facilitated sensitive detection, while calibration achieved robust quantification across a wide dynamic range (50–8000 ppb). Method validation was conducted as per ICH guidelines, demonstrating exceptional performance metrics, including intra-day precision (RSD 95%), and stability under various storage conditions. The study provides a streamlined methodology for pharmaceutical analysis and regulatory compliance, ensuring reliable detection of UDCA in clinical studies.