A novel LC-MS/MS method was developed and validated for determining the concentration of Ritlecitinib in rat plasma, demonstrating simplicity, precision, accuracy, sensitivity, and reproducibility. The process included liquid-liquid extraction using dichloromethane as the organic solvent, followed by chromatographic separation on an Inertsil ODS-3 column (100 mm x 4.6 mm, 3 µm). The mobile phase, composed of acetonitrile and 0.1% formic acid in a 30:70 v/v ratio, was delivered at a flow rate of 1.0 ml/min, achieving a retention time of 2.724 minutes for Ritlecitinib and 2.727 minutes for Ritlecitinib D6. Detection utilized an Electron Spray Ionization interface with the SCIEX QTRAP 5500 LC-MS/MS system in multiple reaction monitoring mode. This approach enabled simultaneous identification of Ritlecitinib and its deuterated form, showing m/z transitions from 286.159 to 105.7458 and 292.1595 to 111.7468, respectively. The method exhibited linearity across a 5.00–100.00 ng/ml concentration range, with a correlation coefficient (r²) of ≥0.99977. Stability was proven through freeze-thaw cycles and short- term and long-term stability studies. The results show that the proposed strategy can be efficiently and effectively used for routine analysis of Ritlectinib in rat plasma.