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Enzalutamide is a potent androgen-signaling receptor inhibitor and is licensed for the treatment of metastatic castration–resistant prostate cancer. N-desmethylenzalutamide is the active metabolite of enzalutamide. A method to quantitate enzalutamide and its active metabolite was developed and validated according to the European Medicine Agency guidelines.Enzalutamide and N-desmethylenzalutamide were extracted by protein precipitation, separated on a C18 column with gradient elution and analyzed with tandem quadrupole mass spectrometry in positive ion mode. A stable deuterated isotope (D6-enzalutamide) was used as an internal standard. The method was tested and stability was studied in real-life patients with metastatic castration–resistant prostate cancer patients treated with enzalutamide.The calibration curve covered the range of 500–50,000 ng/mL. Within- and between-day precisions were <8% and accuracies were within 108% for both enzalutamide and N-desmethylenzalutamide. Precisions for lower limit of quantification level were <10% and accuracies within 116% for enzalutamide and N-desmethylenzalutamide. Enzalutamide and N-desmethylenzalutamide stability was proven for 24 hours for whole blood at ambient temperature and 23 days for plasma at both ambient temperature and 2–8°C. Long-term patient plasma stability was shown for 14 months at −40°C.This bioanalytical method was successfully validated and applied to determine plasma concentrations of enzalutamide and N-desmethylenzalutamide in clinical studies and in routine patient care.