We set a goal of understanding how the multiple components in the fluorometric/colorimetric method may influence the outcome. Addition of NaOH was inevitable because NaBH4 was essential for the palladium-catalyzed allylic C-O bond cleavage under air atmosphere , and NaBH4 must be stabilized by NaOH in water. NaBH4 and NaOH react with NH4OAc, and the resulting basicity influences the stability of NaBH4. NH4OAc was found to retard the reaction because it quenches NaBH4, but it was necessary to avoid acidic conditions in the assay. We titrated multiple components in a combinatorial manner for the first time since we began developing and applying fluorometric or colorimetric methods for palladium. When there was more NaOH than NH4OAc, the excess NaOH turned the solution purple in the absence of palladium. Although the UV-vis spectrum of the purple solution resembled that of resorufin, RAE was not converted to resorufin because neutralization with additional NH4OAc or acid reversed the UV-vis spectrum back to that of RAE. The reversible reaction between RAE and NaOH was further supported by 1 H NMR analysis. Unfortunately, we were unable to determine the structure of the transient product under basic conditions due to its poor solubility. Based on the previously observed mode of reactivity of related resorufin derivatives, we speculate that NaOH undergoes reversible conjugate addition to form keto-1, which may be in equilibrium with its tautomer, enol-1 (Scheme 2). Although the hypothesized mechanism may be intriguing, this is detrimental to achieving the goal of developing a palladium-specific quantification method. Thus, it is important to ensure that the NH4OAc concentration exceeds the NaOH concentration. Our palladium stock solutions were stored in 500 mM HCl, which partially eliminated NaOH in assay solutions. Because of this background reaction identified in the current study, we changed the order of addition of reagents to avoid such an undesired reaction. The stop-and-go strategy is convenient for those who have no access to continuous monitoring of the assay.