The heterogeneous distribution of delivery or treatment modalities within the tumor mass is a crucial limiting factor for a vast range of theranostic applications. Understanding the interactions between a nanomaterial and the tumor microenvironment will help to overcome challenges associated with tumor heterogeneity, as well as the clinical translation of nanotheranostic materials. This study aims to evaluate the influence of protein surface adsorption on gold nanoparticle (GNP) biodistribution using high-resolution computed tomography (CT) preclinical imaging in C57BL/6 mice harboring Lewis lung carcinoma (LLC) tumors. LLC provides a valuable model for study due to its highly heterogenous nature, which makes drug delivery to the tumor challenging. By controlling the adsorption of proteins on the GNP surface, we hypothesize that we can influence the intratumoral distribution pattern and particle retention. We performed an in vitro study to evaluate the uptake of GNPs by LLC cells and an in vivo study to assess and quantify the GNP biodistribution by injecting concentrated GNPs citrate-stabilized or passivated with bovine serum albumin (BSA) intratumorally into LLC solid tumors. Quantitative CT and inductively coupled plasma optical emission spectrometry (ICP-OES) results both confirm the presence of particles in the tumor 9 days post-injection (n = 8 mice/group). A significant difference is highlighted between citrate-GNP and BSA-GNP groups (** p < 0.005, Tukey's multiple comparisons test), confirming that the protein corona of GNPs modifies intratumoral distribution and retention of the particles. In conclusion, our investigations show that the surface passivation of GNPs influences the mechanism of cellular uptake and intratumoral distribution in vivo, highlighting the spatial heterogeneity of the solid tumor.