The stability of baicalin in buffered aqueous solutions at different pHs and in biological fluids, including plasma, urine and tissue homogenates, were investigated in vitro. Structures of the degradation products of baicalin were elucidated by liquid chromatography-electrospray ion trap mass spectrometry and the degradation mechanism was proposed with the aid of electron paramagnetic resonance spectrometry. The results showed that the degradation of baicalin was pH- and temperature-dependent. The oxidation-reduction reaction intermediated by phenoxyl radicals is the major degradation process for baicalin in plasma and urine in vitro, whereas baicalin mainly undergoes hydrolysis and phase II metabolic pathways when spiked in tissue homogenates. It is found that acidification can stabilize baicalin in urine and plasma (the final pH adjusted to 3.0-4.0), and baicalin is relatively stable in tissues homogenized with methanol.