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First bioanalytical method being reported for quantification of tideglusib in mice plasma on LC–MS/MS.Simple sample processing method and shorter run time (3.0 min) enables this method as high throughput assay.Method was validated as per regulatory guidelines.The method is specific, precise, accurate and no matrix effect was observed and linear from 20.2 to 1008 ng/mL.The method was successfully used in a mice pharmacokinetic study and for the first time pharmacokinetic parameters for tideglusib in mice.A sensitive, specific and rapid LC-ESI–MS/MS method has been developed and validated for the quantification of tideglusib in mice plasma using warfarin as an internal standard (I.S.) as per regulatory guidelines. Sample preparation was accomplished through liquid-liquid extraction process. Chromatographic separation was performed on Atlantis dC18 column using mobile phase A (acetonitrile) and B (5 mM ammonium acetate in water) in a flow-gradient mode. Elution of tideglusib and the I.S. occurred at ˜2.06 and 1.29 min, respectively. The total chromatographic run time was 3.2 min. A linear response function was established in the concentration range of 20.2–1008 ng/mL. The intra- and inter-day accuracy and precision were in the range of 4.61–12.6 and 6.04–11.8%, respectively. This novel method has been applied to a pharmacokinetic study in mice.