Cinnamylideneacetophenones have been extensively used as versatile starting materials in numerous different transformations. The structural characterization of this type of compounds is, therefore, of crucial importance since it can give information on the chemistry, reactivity and also the potential biological activity of this type of compounds. Thus, 24 derivatives were systematically studied by tandem mass spectrometry (MS2) with electrospray ionization (ESI), in positive ion mode. The protonated molecules, [M + H]+, formed under ESI conditions were induced to dissociate and the fragmentation patterns were studied. The information collected provided important structural information on the type of substituents present and constitute an important database concerning this family of compounds. Overall, it was found that the substitution pattern of the cinnamylideneacetophenone derivatives changes the ESI-MS2 fragmentation considerably. These results indicate that ESI-MS2 is a useful technique for distinguishing positional isomers of these cinnamylideneacetophenone derivatives. Copyright © 2011 John Wiley & Sons, Ltd.