Plant organ detachment occurs through the activation of specific, highly specialized cells, named the abscission zone (AZ). Molecular and biochemical transformations are induced in this specific tissue region that lead to the loosening of the cell walls, middle lamellae dissolution, and, as a consequence, the separation of the organ from the maternal plant. Understanding the mechanisms of AZ activation requires elucidating changes in the composition of cell walls following abscission. In this chapter, we present a protocol developed for flower lupine AZ analyses that can be used to study the AZs of different organs formed in various species. The procedure outlines the proper excision of biological material, particularly the successful AZ collection and the preparation for cross-sectioning. Furthermore, tissue fixation, dehydration, saturation with resin, and sectioning are thoroughly described. A detailed methodology for the immunocytochemical reactions using antibodies to perform a dot-blot assay, as well as the localization of pectins, extensins, and hemicelluloses in the AZ with the fluorescence microscope is provided. This comprehensive protocol provides a basis to elucidate which cell wall components are modified to determine the timing of organ separation.