Quantitative analysis using a turn-on fluorescent probe is inherently difficult due to the dependency of the fluorescence intensity on the probe concentration. To overcome this limitation, we developed an in situ quantification method using a turn-on fluorescent probe and a standard fluorophore, which are colocalized by protein tag technology. This protocol describes the synthesis of a Zn2+ probe, named ZnDA-1H, and the procedure to quantify the labile Zn2+ concentration in the Golgi of live HeLa cells by confocal fluorescence microscopy. For complete details on the use and execution of this protocol, please refer to Kowada et al. (2020).