The present study outlines the useability of two plant O-methyl transferases (O-MTs), phenylpropanoid and flavonoid O-methyl transferase (PFOMT) and soy O-methyl transferase (SOMT-2), of classes I and II for the biocatalytic methylation of common structural motifs encountered throughout the group of in plant polyphenolic compounds. PFOMT was biophysically characterized through and the solution of a novel crystal structure of its apo-form. In vivo studies using SOMT-2 showed its capability to methylate flavonoids and stilbenes at the 4'-position. It was demonstrated that the activity of class I plant O-MT, PFOMT, could be modulated by pH and magnesium concentration to achieve previously unobserved methylations of non-catecholic moieties. Additionally, a systematic grid of 15 flavonoids with different substitutions at the B-ring was characterized by tandem mass-spectrometry (MS/MS) studies through higher-energy collisional dissociation (HCD) and collision induced dissociation (CID).