Cell-based assays are essential in both basic research and drug discovery. Three-dimensional cellular spheroids are more realistic models of tumors and healthy tissues compared to standard two-dimensional cultures. Employing spheroids improves the reliability and the physiological significance of cell-based assays. We present a detailed drug assay protocol performed with live cellular spheroids. We employ automated epifluorescence live microscopy to investigate the effects of drugs on the spheroids over several days. We describe the spheroid preparation, manipulation, live fluorescence imaging, and data processing. We quantify the autophagy-triggering effects of the drugs (-)-Gossypol and Rapamycin in glioma cell spheroids. The formation of the autophagosomes and the fusion of the autophagosomes with lysosomes in the treated spheroids are monitored over time and space with a mRFP:GFP:LC3 fusion protein.