Abstract Oxidation of natural flavonolignan silybin in an aprotic environment was studied by electrochemical methods, electron paramagnetic resonance (EPR), UV–vis and IR in situ spectroelectrochemistry and chromatographic techniques. The presence of electrochemically generated silybin radical was proven using in situ EPR spectroelectrochemistry by spin trapping method with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO) as a spin trap reagent. The adduct of the silybin radical with BMPO was additionally unambiguously identified by UPLC-ESI-MS/MS. For the first time, the flavonolignan radical was detected upon electrochemical oxidation under such conditions. The one-electron oxidation of silybin leads to a hydroxylated silybin derivative. Its specific redox behaviour can reflect its bioefficiency.