Posttranslational modification of histones during the cell cycle is a major process controlling many aspects of cell division. Among the variety of histone modifications, mitotic phosphorylation of histone H3 at serine 10 (H3S10ph) plays a crucial role, particularly in proper chromosome segregation. Here we aimed at precisely quantifying this phosphorylation dynamics during mitosis in plant cells in order to reveal molecular pathways involved in this process. We describe an analysis pipeline based on 3D image analysis that allows to semiautomatically quantify H3S10ph in mitotic Arabidopsis root cells. We also developed a new method for the compensation of signal attenuation in Z, based on measurement of objects of interest themselves. We show that this new attenuation correction method allows significant gains in accuracy and statistical power. Using this pipeline, we were able to reveal small H3S10ph differences between cells treated with hesperadin, an inhibitor of an H3S10ph kinase, or between Arabidopsis mutants affected in PP2A phosphatase activity. This tool opens new avenues to explore such regulatory pathways in plants, using the wealth of genetic materials available in Arabidopsis. It can also be applied to study other histone posttranslational modifications and more generally to any discrete 3D signals.