OBJECTIVE： To establish a method for the separation and detection of related substances in baicalein， identify its structure and preliminarily explore the degradation mechanism. METHODS： HPLC was adopted to detect the baicalein， related impurities and forced destruction of degradation products in synthesis process： the column was ES Industries® FluoroSep-RP Phenyl with mobile phase of 0.3％ formic acid-methanol-acetonitrile （gradient elution） at a flow rate of 1.0 mL/min， the detection wavelength was 275 nm， the column temperature was 10 ℃，and the injection volume was 10 μL. LC-MS/MS was conducted to identify the related substances and conjecture degradation mechanism： the column was ES Industries® FluoroSep-RP Phenyl with mobile phase of 0.3％ formic acid - methanol  （gradient elution） at a flow rate of 1.0 mL/min， the detection wavelength was 275 nm， column temperature was 10 ℃， and the injection volume was 10 μL； ion source was electrospray ion source， positive and negative ions， nebulizer pressure was 55 psi and the drying gas flow was 11 L/min， drying gas temperature was 350 ℃， capillary voltage was 4.0 kV， detection modes were full-scan first-order MS and selective ion full-scan second-order MS， scan ranges were m/z 100-1 000 （first-order MS） and 50-500 （second-order MS）， ionization voltage was 80-135 eV， and the collision energy was 10-30 eV. RESULTS： The linear range of baicalein was 2.4-480 μg/mL（r＝0.999 9）； RSDs of precision， stability and reproducibility tests were lower than 2.0％； the limit of quantitation was 7.2 ng，the limit of detection was 2.4 ng. Baicalein was well separated with related substance and 3 major degradation products， the related substance was chemical synthesis precursor wood butterfly； the degradation products were 6， 7-quinone derivatives and 7， 8-quinone derivatives， which were isomers； oxidative degradation products were benzoic acid phenyl ester derivatives. CONCLUSIONS： The main mechanisms of alkali degradation and oxidative degradation of baicalein include pyran， reciprocal rearrangement and oxidation reaction； the established method is specific and sensitive， and can be used for the detection of related substances in baicalein.