Abstract: There is an increasing interest in using three-dimensional (3D) cell structures for modeling tumors, organs, and tissue to accelerate translation research. Significant progress has been made in formation of such structures to recapitulate the in vivo environment but performing complex assays with them can be challenging. We present here results from automated organoid assays using a Pu·MA System coupled with high content imaging and fast fluorescence kinetic read-outs. The flowchip contains organoid sample wells connected to multiple reservoirs that can contain various assay reagents. Each organoid is located in a protective chamber and fluids are transferred in and out from the sides using pneumatic pressures. This allows media exchange, sample staining, wash steps, and other processing all to be performed without disruption to, or loss of 3D sample. The bottom of the sample chamber is thin optically clear plastic compatible with high resolution fluorescence imaging. The whole system can be kept in an incubator allowing long term cellular assays to be performed. The system has been used for applications in the areas of long-term toxicity, oncology therapeutics, single organoid secretion, and metabolite sampling. In the first example, we automated a protocol for assaying cytotoxicity effects of anti-cancer drugs on cancer spheroids. Spheroids were formed with HeLa or HepG2 cells then pre-loaded into a flowchip reservoirs along with compound solutions. The flowchips were placed into a Pu·MA System and incubated for 48 hours. Next, media was removed and spheroids were stained with viability dyes. The spheroids were imaged and analyzed by ImageXpress Micro Confocal Imaging System. Confocal imaging allowed 3D resolution of spheroid structures and complex analysis of cytotoxic effects. In addition, conditioned media was removed and analyzed for VEGF. A concentration dependent decrease in VEGF secretion was observed with HepG2 cells. In the second application the flow-chip system was used for the functional evaluation of calcium oscillations in neurospheroids. Neural 3D cell spheroids (microBrain, Stemonix) were incubated with various neuro-active compounds and neuronal activity, or “firing” was assessed using calcium sensitive dyes and fast kinetic fluorescence imaging (FLIPR). The Pu·MA System automated multiple additions of compounds to the neurospheroids and allowed evaluation of concentration-responses and other effects. Kinetic fluorescence patterns were recorded by FLIPR directly in the flowchips associated with “firing” of neuronal co-culture spheroids. Observed perturbations in the calcium oscillations patterns were consistent with the mechanism of action of the compounds. This novel assay method using microfluidics enables automation of 3D cell-based cultures that mimic in vivo conditions, performs multi-dosing protocols and multiple media exchanges, provides gentle and convenient handling of spheroids and organoids, and allows a wide range of assay detection modalities.