Abstract: In recent years, researchers have transitioned from traditional 2D assays to more complex 3D cell models, as they are shown to recapitulate the in vivo environment and serve as a more predictive tool for drug discovery. One area gaining attention is the use of patient-derived organoids (PDO) for oncology research. PDOs are generated from tumor biopsies and can serve as models to understand tumor growth and resistance to therapies. With this transition, there has been an increased need to automate the processing and analysis of 3D cell culture assays for a more simplified, “hands-off” workflow that yields better results. We present here results from semi-automated PDO assays using a Pu·MA System coupled with high content imaging. 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 application presented here, PDOs from a triple negative breast cancer (TNBC) patient were grown and transferred into flowchips. PDOs were incubated in the presence of anti-cancer compounds for 24 – 48 hours and then stained for either viability markers or E-cadherin/CD44 markers. The use of flowchips for automated fluid exchange helped protect the sensitive structures from damage and perform automated processing of organoids. High resolution images of PDOs were obtained using ImageXpress Micro Confocal system and analyzed with MetaXpress software. Resistance of PDOs to chemotherapeutic agents was observed consistent with tumor response in the patient. 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.