In vitro cytotoxicity of three (E)-3-(4′-X-benzylidene)-1-indanones (2a-c) displayed lower cytotoxicity towards murine P388 and L1210 leukemic cells as well as human Molt 4/C8 and CEM T-lymphocytes than the respective six- (3a-c) and seven-membered (4a-c) analogs. To study whether thiol reactivity—as a possible basis of their mechanism of action—correlates with the observed cytotoxicities, kinetics of the non-enzyme catalyzed reactions with reduced glutathione (GSH) and N-acetylcysteine (NAC) of 2a-c were investigated. Furthermore, it was also the aim of the work to compare the thiol reactivity of the open-chain chalcones (1) and their carbocyclic analogs (2-4) with different ring sizes (n = 5–7). The reactivity of the compounds and the stereochemical outcome of the reactions were evaluated using high-pressure liquid chromatography–mass spectrometry (HPLC-MS). Molecular modeling calculations were performed to rationalize the high initial rate and low conversion of the 2a indanone in comparison with those of the carbocyclic analog tetralone (3a) and benzosuberone (4a). Thiol reactivity and cancer cell cytotoxicity showed a dependence on both the ring size and the nature of aromatic substituents.