Abstract: This study established and validated an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analytical method for the simultaneous determination of plasma pharmacokinetic characteristics and major-organ tissue distribution profiles of five key active components, namely geniposide, liquiritin, glycyrrhizic acid, berberine, and phellodendrine, from Zhizi Baipi Decoction (ZBD) in rat models with adjuvant arthritis (AA). After ZBD was intragastrically administered to AA rats, plasma samples as well as tissue samples of major organs including the heart, liver, spleen, lung, and kidney were collected at multiple time points. After the completion of systematic methodological validation, the five key active components mentioned above were quantitatively determined to characterize their specific pharmacokinetic parameters in plasma and time-dependent changes in tissue distribution patterns. The results of method validation demonstrated that the specificity, linearity, accuracy, precision, stability, extraction recovery, and matrix effect of the established UPLC-MS/MS method all fully satisfied the relevant technical requirements for biological sample analysis. Among the five active components, phellodendrine achieved the maximum peak concentration (C_max) in plasma of 1 019.31 μg/L, while geniposide had the shortest peak time (T_max) and the longest half-life (t_1/2) of 8.42 h, which was markedly longer than those of the other four compounds. In addition, the plasma concentration-time curves of glycyrrhizic acid, berberine, and phellodendrine all presented a distinct double-peak phenomenon, which suggested that their absorption and metabolism processes in vivo may involve multiple pathways. Meanwhile, tissue distribution analysis showed that geniposide predominantly accumulated in the kidney, liver, and spleen, with the order of kidney > liver > spleen > heart > lung. Liquiritin, glycyrrhizic acid and berberine were all mainly enriched in the liver and kidney, indicating that these two organs are the main sites for the accumulation of the three components. Phellodendrine attained maximum concentrations in the liver and kidney, and its concentration in the kidney was similar to that in the spleen, lung, and heart. The UPLC-MS/MS method established in this study is sensitive, accurate and stable, and suitable for the simultaneous exploration of the distribution trajectories and accumulation patterns of multiple ZBD constituents in the plasma and various tissues of AA model rats. This study provides a reliable experimental basis for clarifying the pharmacodynamic material basis of ZBD and guiding its rational clinical application in the treatment of related diseases.