We kinetically studied the reaction between endogenous thiols such as -γ-glutamylcysteine (γ-Glu-Cys), cysteine (Cys), glutathione (GSH), homocysteine (Hcy), cysteinylglycine (Cys-Gly), and dihydrolipoic acid (DHLA)- with (E)-2-(benzo[d]thiazol-2-yl)-3-(4-morpholinophenyl)acrylonitrile (JGB). Studies conducted by NMR and ESI-MS/MS have demonstrated that this reaction occurs via thiol-addition toward the double bond present in JGB. Considering the product analysis and the pH-dependence of the second order rate constant (kN), we proposed a mechanism that involves the rapid protonation of JGB giving place to an intermediate following by the thiolate attack yielding a final product. Moreover, this probe could successfully sense thiols in the human neuroblastoma SH-SY5Y cells.