Studying phenotypic variation in plant pathogenesis provides fundamental information about the nature of disease resistance. Cellular mechanisms that alter pathogenesis can be elucidated with confocal microscopy, but systematic phenotyping platforms—from sample processing to image analysis—to investigate this do not exist. We have developed a platform for 3D phenotyping of cellular features underlying variation in disease development by fluorescence-specific resolution of host and pathogen interactions across time (4D). A confocal microscopy phenotyping platform compatible with different maize-fungal pathosystems (fungi: Setosphaeria turcica, Cochliobolus heterostrophus, and Cercospora zeae-maydis) was developed. Protocols and techniques were standardized for sample fixation, optical clearing, species-specific combinatorial fluorescence staining, multi-sample imaging, and image processing for investigation at the macro scale. The sample preparation methods presented here overcome challenges to fluorescence imaging such as specimen thickness and topography as well as physiological characteristics of the samples such as tissue autofluorescence and presence of cuticle. The resulting imaging techniques provide interesting qualitative and quantification information not possible with conventional light or electron 2D imaging.