This chapter outlines detailed methods and protocols for studying the structure and mechanisms of abscission layers (ALs) in rice. Utilizing rice spikelets as a primary example, these protocols provide comprehensive techniques for analyzing AL development, including tissue preparation, microscopy, and histochemical assays. The AL is a specialized tissue where cell separation takes place, and it is crucial for processes such as organ shedding and fruit drop. By examining the AL, researchers can uncover the physiological and genetic factors governing plant organ separation. These insights are pertinent for advancing agricultural practices and crop improvement, as understanding the dynamics of the AL can lead to the development of rice varieties with enhanced traits related to abscission and grain retention. These improvements can result in better yield stability and reduced post-harvest losses, which are essential for meeting the food demands of a growing global population. By focusing on the genetic and physiological mechanisms governing the AL, researchers can develop innovative strategies to optimize rice production and contribute to food security.