Catechols are an important class of analytes occurring in many natural and synthetic products. Electrospray ionization in negative mode is the preferred way of ion generation for these compounds; however, studies in positive ion mode can reveal their potential for in-source oxidation and further structural changes, some of which may also occur in the solution phase. Therefore in-source oxidation can provide a forward look into the potential for solution oxidation. 1:1 Acetonitrile/water solutions of catechol (Cat), 4,5-dichlorocatechol (4,5-DCC), 3,4-dichlorocatechol (3,4-DCC) and tetrachlorocatechol (TCC) were analyzed by positive ion ultrahigh-performance liquid chromatography (UHPLC/ESI-MS) and UHPLC/ESI-MS/MS under various emitter voltages to assess their liability towards in-source oxidation. Structural information for in-source generated compounds was obtained through the use of product ion scans. Using catechols as probe compounds, we have demonstrated that under the conditions used in many analytical laboratories in-source oxidation can severely affect the sensitivity and response functions of an analyte. Under standard UHPLC conditions (300 μL/min flow rate), Cat, 3,4-DCC, 4,5-DCC and TCC can undergo in-source oxidation. The extent of oxidation is dependent either on the instrument or on the characteristics of the emitter. This is evident by a change in the isotopic pattern of these compounds and the generation of ions at lower m/z values due to a loss of 1 and/or 2 hydrogens and electrons. In the case of catechol, the formation of a dimer resulting from in-source oxidation reactions was observed. This dimer has the same fragmentation pattern as the dimer generated by oxidation in the solution phase. The present work demonstrates the potential of positive ion ESI for oxidizing electroactive compounds during regular analytical operation using commercially available mass spectrometers. Using Cat and some of its chlorinated analogues as probe compounds, we have demonstrated that under the conditions used in many analytical laboratories in-source oxidation and dimerization can and does take place.