Enzalutamide was recently approved for the treatment of castration-resistant prostate cancer. In this study, the related substances in enzalutamide bulk substance were analyzed qualitatively and quantitatively. Four degradation products (Oxi, A9, P1 and P2) of enzalutamide were isolated using semi-preparative liquid chromatography and characterized using nuclear magnetic resonance and mass spectrometry. In addition, two reversed-phase liquid chromatography methods were developed for quantification of 13 potential impurities and the main constituent, respectively. These two methods were validated and applied during the impurity studies and quality control analysis of the laboratory-prepared samples of enzalutamide.