High throughput (HT) standardized method for generation, imaging and analysis of tumor spheroids. (a) Ultra-low attachment (ULA) 96-well round-bottomed plates were used to generate suspension cultures of reproducibly sized, single spheroids in each well. (b) Fully automated imaging and analysis was carried out on a Celigo cytometer using the Colony Counting Embryoid Body application with 1/16 field of view/well. A screen print shows reproducibly sized U-87 MG spheroids across the plate and detail of the segmentation analysis around the spheroid. Scale bar: 500 μm. (c) U-87 MG tumor spheroid growth curve. Values are means ± SD (n = 6). Representative images obtained on Celigo for each timepoint are shown. Scale bar 500 μm. (d) Frequency plots of 4-day U-87 MG spheroid volume (μm3 × 107) across a plate containing 60 spheroids.