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First LC–MS/MS method being reported for the simultaneous quantification of darolutamide and ORM-15341 in mice plasma.Simple sample processing method and shorter run time (2.5 min) enables this method as a high throughput assay.Method was validated as per regulatory guidelines.The method is specific, precise, accurate and no matrix effect was observed.Method was used in a mice pharmacokinetic study to derive the pharmacokinetic parameters for analytes.A sensitive and rapid LC–MS/MS method was developed and validated for the simultaneous quantitation of darolutamide and its active metabolite i.e. ORM-15341 in 50 μL mice plasma using bicalutamide as an internal standard (I.S.) as per regulatory guidelines. Sample processing was accomplished through liquid-liquid extraction. Chromatographic separation was achieved using an Atlantis C18 column with an isocratic mobile phase comprising 0.2% formic acid:acetonitrile (35:65, v/v) at a flow rate of 0.8 mL/min within 2.5 min. Detection and quantitation were done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 397 → 202, 395 → 202 and 429 → 255 for darolutamide, ORM-15341 and I.S, respectively in the negative ionization mode. The calibration curve was linear from 0.61–1097 ng/mL for both darolutamide and ORM-15341. The intra- and inter-day precisions were in the range of 1.34–13.8 and 4.85–12.9 and 3.91–13.7 and 6.54–14.2%, for darolutamide and ORM-15341, respectively. Darolutamide and ORM-15341 were found to be stable under different stability conditions. The validated method was applied to a pharmacokinetic study in mice.