MotivationQuantitative descriptions of multi-cellular structures from optical microscopy imaging are prime to understand the variety of three-dimensional (3D) shapes in living organisms. Experimental models of vertebrates, invertebrates and plants, such as zebrafish, killifish,DrosophilaorMarchantia, mainly comprise multilayer tissues, and even if microscopes can reach the needed depth, their geometry hinders the selection and subsequent analysis of the optical volumes of interest. Computational tools to “peel” tissues by removing specific layers and reducing 3D volume into planar images, can critically improve visualization and analysis.ResultsWe developed VolumePeeler, a versatile FIJI plugin for virtual 3D “peeling” of image stacks. The plugin implements spherical and spline surface projections. We applied VolumePeeler to perform peeling in 3D images of spherical embryos, as well as non-spherical tissue layers. The produced images improve the 3D volume visualization and enable analysis and quantification of geometrically challenging microscopy datasets.AvailabilityImageJ/FIJI software, source code, examples, and tutorials are openly available inhttps://cimt.uchile.cl/mcerdaSupplementary InformationThe online version contains supplementary material available at 10.1186/s12859-023-05403-z.