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Despite the vast potential of nanoparticles in anticancer therapies, the method of evaluating nanoparticle penetration within specific tumors is neither standardized nor mainstream. Multicellular tumor spheroids have been established as a popular model for in vitro screening of nanoparticles; however, their utility would be expanded considerably by improved quantification of nanoparticle diffusivity. This paper outlines a method to accurately estimate the diffusivity of nanoparticles in multicellular tumor spheroids using an unsteady-state diffusion based simulation model. This simulation model has been shown to accurately estimate diffusivity using synthetic data. Validation of the model's use in drug-delivery studies is accomplished through the high throughput creation of consistent tumor spheroids, introduction of fluorescently-labeled nanoparticles into the engineered tumors, and image processing of spheroid samples. Once validated, this system will allow researchers to efficiently quantify diffusivity for their nanoparticle drug delivery systems.