Auxin plays a critical role in the inhibition of organ abscission. The changing of auxin gradient across the abscission zone (AZ) is the primary determinant of the abscission initiation and activation. To better understand the role of auxin, the distribution and content of auxin at different developmental stages of AZ need to be determined. This protocol provides a convenient and efficient procedure for precise localization of auxin in the petal AZ of rose (Rosa hybrida). It can be combined with plant hormone metabolomics to more accurately reflect changes in auxin during the abscission process. This method serves as a powerful tool for auxin detection and can be applied to broader plant hormone research.