Patient-derived 3D organoids show great promise for understanding patient heterogeneity and chemotherapy response in human-derived tissue. The combination of organoid culture techniques with mass spectrometry imaging provide a label-free methodology for characterizing drug penetration, patient-specific response, and drug biotransformation. However, current methods used to grow tumor organoids employ extracellular matrices which can produce small molecule background signal during mass spectrometry imaging analysis. Here, we develop a method to isolate 3D human tumor organoids out of a Matrigel extracellular matrix into gelatin mass spectrometry compatible microarrays for high-throughput MS imaging analysis. The alignment of multiple organoids in the same z-axis is essential for sectioning organoids together and for maintaining reproducible sample preparation on a single glass slide for up to hundreds of organoids. This method successfully removes organoids from extracellular matrix interference and provides an organized array for high-throughput imaging analysis to easily identify organoids by eye for area selection and further analysis. With this method, mass spectrometry imaging can be readily applied to organoid systems for pre-clinical drug development and personalized medicine research initiatives.