Organotypic spheroids from malignant glioma resemble the biological complexity of the original tumor and are therefore appealing to study anticancer drug responses. Accurate and reproducible quantification of response effect has been lacking to determine drug responses in this three-dimensional tumor model. Lactate dehydrogenase (LDH) activity was demonstrated in cryostat sections of spheroids using the tetrazolium salt method. Calibrated digital image acquisition of the stained cryostat sections enables quantification of LDH activity. Fully automated image cytometry reliably demarcates LDH-active and LDH-inactive tissue areas by thresholding at specific absorbance values. The viability index (VI) was calculated as ratio of LDH-active areas and total spheroid tissue areas. Duplicate staining and processing on the same tissue showed good correlation and therefore reproducibility. Sodium azide incubation of spheroids induced reduction in VI to almost zero. We conclude that quantification of viability in cryostat sections of organotypic multicellular spheroids from malignant glioma can be performed reliably and reproducibly with this approach.