Tafamidis is a first-in-class inhibitor of transthyretin amyloid fibril formation. It has been available in Argentina, Japan, and Mexico for the treatment of transthyretin amyloidosis in adult patients with early-stage symptomatic polyneuropathy. In this study, a rapid and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the assay of tafamidis in rat plasma. The method was also assessed for its applicability to pharmacokinetic studies in rats. Tafamidis was extracted from rat plasma by the liquid-liquid extraction method using hydrochloric acid and ethyl acetate. A reversed-phase C18 column and a mobile phase consisting of 10 mM ammonium formate and acetonitrile were used to achieve chromatographic separation. The flow rate for the mobile phase was set at 0.3 mL/min. Tafamidis and 2-CBC, which was used as the internal standard (IS), were analyzed by multiple reaction monitoring in negative ESI mode at m/z transitions of 305.4 → 261.4 for tafamidis and 271.7 → 227.8 for the IS. The lower limit of quantification of tafamidis was obtained as 3 ng/mL, and the calibration curve was linear over a concentration range of 3–3000 ng/mL (R2 > 0.99). The validation parameters investigated, which were specificity, precision, accuracy, matrix effect, recovery, and stability, were well within acceptable limits. The method was successfully used for the evaluation of the pharmacokinetics of tafamidis in rats.