Electrochemical oxidation of peptides and proteins has been shown to lead to specific cleavage next to tyrosine (Tyr) and tryptophan (Trp) residues which makes the coupling of electrochemistry to mass spectrometry (EC-MS) a potential instrumental alternative to chemical and enzymatic cleavage. A set of Tyr and Trp-containing tripeptides has been studied to investigate the mechanistic aspects of electrochemical oxidation and the subsequent chemical reactions including peptide bond cleavage, making this the first detailed study of the electrochemistry of Trp-containing peptides. The effect of adjacent amino acids was studied leading to the conclusion that the ratios of oxidation and cleavage products are peptide-dependent and that the adjacent amino acid can influence the secondary chemical reactions occurring after the initial oxidation step. The effect of parameters such as potential and solvent conditions showed that control of the oxidation potential is crucial to avoid dimer formation for Tyr and an increasing number of oxygen insertions (hydroxylations) for Trp, which occur above 1000 mV (vs Pd/H(2)). While the formation of reactive intermediates after the first oxidation step is not strongly dependent on experimental conditions, an acidic pH is required for good cleavage yields. Working under strongly acidic conditions (pH 1.9-3.1) led to optimal cleavage yields (40-80%), whereas no or little cleavage occurred under basic conditions. Online EC-MS allowed determining the optimal potential for maximum cleavage yields, whereas EC-LC-MS/MS revealed the nature and distribution of the reaction products.