Immunofluorescent approach uses fluorophore-conjugated antibodies to target molecules of interest for the observation of their distribution and microenvironment at cellular or tissue level. In connection with modern fluorescence microscopy, immunofluorescence has high sensitivity and specificity, resulting in a wide range of applications in animal studies. However, the protocols of immunofluorescence are seldom reported. To this end, we describe an optimized protocol for the detection of plant hormone abscisic acid (ABA) in monocot crop species, including maize and rice. By using immunofluorescence technique provided from this protocol, investigation of ABA distribution will continue to deeper our insights in crop developments and stress responses.