The invention discloses a method of detecting and separating related substances in pomalidomide or an oral solid preparation thereof through high performance liquid chromatography (HPLC) in content, wherein the method includes the following steps: (1) preparing a system suitability solution; (2) carrying out a system suitability test; (3) preparing a to-be-detected solution; and (4) performing detection according to the following HPLC conditions: a mobile phase, wherein gradient elution is carried out with an ion pair phosphoric acid buffer liquid-acetonitrile as a mobile phase A and methanol-acetonitrile as a mobile phase B; an ultraviolet detector, wherein detection wavelength is 200-400 nm; a chromatographic column, wherein octadecylsilane chemically bonded silica is employed as a filler; flow velocity is 0.5-2.0 ml/min; and column temperature is 10-60 DEG C. The method solves the problem of detecting and separating the related substances in pomalidomide or the oral solid preparation thereof.