Abstract BACKGROUND Medulloblastoma is the most common malignant pediatric brain tumor, originating from the cerebellum, a part of the brain that controls balance, coordinated movement and other non-motor behaviors. 2D cell cultures are commonly used for testing of new compounds in high throughput screening (HTS) targeting this cancer, largely because there are numerous highly optimized assays available for analyzing this cell culture model. However, they have significant limitation in mimicking in vivo conditions of the tumor microenvironment. 3D cell-culture methods have been embraced to enhance the modeling of in vivo tissue, replicating the multicellular architecture and the characteristics of the extracellular matrix. Presently, there are efforts to integrate 3D cell-culture with HTS to yield more clinically relevant results. However, one of the requirements for this is a 3D-compatible cell-based assay suitable for HTS. We developed such an assay which measures the activity of YAP/TAZ, a central molecule of Hippo-signaling pathway, and has been implicated in medulloblastoma. It plays a crucial role in maintaining tissue balance by coordinating proliferation, apoptosis, and differentiation. METHODS We cloned 8xGTIIC-NlucP, a brighter and more sensitive luciferase (Nanoluciferase) reporter plasmid that detects YAP/TAZ activity. Responsiveness to YAP/TAZ activity, and its inhibition were detected using a dual luciferase assay in the medulloblastoma cell lines after transfection. A normalization plasmid with a constitutive promoter for firefly luciferase was used in this assay to differentiate compounds that affect YAP/TAZ activity specifically as opposed to those that are toxic to the cells. RESULTS A robust signal for YAP/TAZ activity, and its reduction when cells were treated with inhibitor, was seen in the three medulloblastoma cell lines used, which further validates the result. CONCLUSION In conclusion, we were able to develop a 3D-compatible cell-based assay with Nanoluciferase which can be a foundation for HTS targeting medulloblastoma using 3D cultures.