Tissue-clearing methods allow every cell in the mouse brain to be imaged without physical sectioning. However, the computational tools currently available for cell quantification in cleared tissue images have been limited to counting sparse cell populations in stereotypical mice. Here, we introduce NuMorph, a group of analysis tools to quantify all nuclei and nuclear markers within the mouse cortex after clearing and imaging by light-sheet microscopy. We apply NuMorph to investigate two distinct mouse models: a Topoisomerase 1 (Top1) model with severe neurodegenerative deficits and a Neurofibromin 1 (Nf1) model with a more subtle brain overgrowth phenotype. In each case, we identify differential effects of gene deletion on individual cell-type counts and distribution across cortical regions that manifest as alterations of gross brain morphology. These results underline the value of whole-brain imaging approaches, and the tools are widely applicable for studying brain structure phenotypes at cellular resolution. Display omitted •NuMorph accurately quantifies all cortical nuclei and cell-type-specific markers•Top1 deletion results in increased neuronal degeneration in frontal areas•Nf1 deletion leads to structural changes that recapitulate human MRI findings•Structural changes are driven by increased gliogenesis in the Nf1 knockout model Krupa et al. develop an image analysis toolbox called NuMorph that accurately quantifies all nuclei within the mouse cortex after tissue clearing and light-sheet imaging. They implement NuMorph to characterize region- and cell-type-specific deficits present in two distinct mouse models, demonstrating the advantages of a whole-brain imaging approach.