© 2016, Biomedical Engineering Society. A preclinical testing model for cancer therapeutics that replicates in vivo physiology is needed to accurately describe drug delivery and efficacy prior to clinical trials. To develop an in vitro model of breast cancer that mimics in vivo drug/nutrient delivery as well as physiological size and bio-composition, it is essential to describe the mass transport quantitatively. The objective of the present study was to develop in vitro and computational models to measure mass transport from a perfusion system into a 3D extracellular matrix (ECM). A perfusion-flow bioreactor system was used to control and quantify the mass transport of a macromolecule within an ECM hydrogel with embedded through-channels. The material properties, fluid mechanics, and structure of the construct quantified in the in vitro model were input into, and served as validation of, the computational fluid dynamics (CFD) simulation. Results showed that advection and diffusion played a complementary role in mass transport. As the CFD simulation becomes more complex with embedded blood vessels and cancer cells, it will become more recapitulative of in vivo breast cancers. This study is a step toward development of a preclinical testing platform that will be more predictive of patient response to therapeutics than two-dimensional cell culture.