BackgroundIt has been proposed that engineering the C4photosynthetic pathway into C3crops could significantly increase yield. This goal requires an increase in the chloroplast compartment of bundle sheath cells in C3species. To facilitate large-scale testing of candidate regulators of chloroplast development in the rice bundle sheath, a simple and robust method to phenotype this tissue in C3species is required.ResultsWe established a leaf ablation method to accelerate phenotyping of rice bundle sheath cells. The bundle sheath cells and chloroplasts were visualized using light and confocal laser microscopy. Bundle sheath cell dimensions, chloroplast area and chloroplast number per cell were measured from the images obtained by confocal laser microscopy. Bundle sheath cell dimensions of maize were also measured and compared with rice. Our data show that bundle sheath width but not length significantly differed between C3rice and C4maize. Comparison of paradermal versus transverse bundle sheath cell width indicated that bundle sheath cells were intact after leaf ablation. Moreover, comparisons of planar chloroplast areas and chloroplast numbers per bundle sheath cell between wild-type and transgenic rice lines expressing the maizeGOLDEN-2(ZmG2) showed that the leaf ablation method allowed differences in chloroplast parameters to be detected.ConclusionsLeaf ablation is a simple approach to accessing bundle sheath cell files in C3species. We show that this method is suitable for obtaining parameters associated with bundle sheath cell size, chloroplast area and chloroplast number per cell.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13007-023-01041-x.