Nanoparticle and other drug delivery platforms have demonstrated promising potential for the delivery of therapeutics or imaging agents in a specific and targeted manner. While a variety of drug delivery platforms have been applied to medicine, in vitro and in silico optimization and validation of these targeting constructs needs to be conducted to maximize in vivo delivery and efficacy. Here, we describe the mathematical and experimental models to predict and validate the transport of a peptide targeting construct through a mock tissue environment to specifically target tumor cells, relative to non-tumor cells. We provide methods to visualize and analyze fluorescence microscopy images, and also describe the methods for creating a finite element model (FEM) that validates important parameters of this experimental system. By comparing and contrasting mathematical modeling results with experimental results, important information can be imparted to the design and functionality of the targeting construct. This information will help to optimize construct design for future therapeutic delivery applications.