The 3-D Spheroid Fluorometric Proliferation/Viability Assay provides a useful tool for modeling tumor response in vitro. The kit utilizes a 3-D Culture Qualified 96 Well Spheroid Formation Plate alongside a specialized Spheroid Formation ECM to drive aggregation and/or spheroid formation of cells. Upon completion of spheroid formation, the spheroid may be treated with pharmacological agents to evaluate tumor viability after drug treatment. Tumor spheroid expansion is visualized microscopically and can be quantitated through image analysis software for real-time and label free evaluation. At the conclusion of the assay, cell viability may be assessed by fluorescence using Resazurin. The 3-D Spheroid Fluorometric Proliferation/Viability Assay offers an in vitro, standardized, three-dimensional, high content format for inducing multicellular tumor spheroid (MCTS) formation and quantitating cell viability within the spheroids in response to pharmacological treatment.