Effective nanoparticle drug delivery to tumor cells typically relies on prolonged systemic circulation of the nanoparticles to allow for extravasation and accumulation in tumor tissue, as well as targeting ligands on the nanoparticles that can mediate receptor-specific uptake by target tumor cells. Due to the ability of polyethylene glycol (PEG) to effectively reduce nonspecific protein binding and cell clearance, PEGylation has become a commonplace strategy for formulating long-circulating nanoparticle systems. However, the precise characteristics (e.g., PEG molecular weight and density) that influence the interactions between PEG-coated nanoparticles and phagocytic immune cells remain poorly understood for many nanoparticle systems, and findings from human studies suggest that the body is further able to mount PEG-specific humoral responses to PEG-coated agents. Additionally, the presence of targeting ligands on the nanoparticle surface may also compromise the extended circulation profile of PEG-coated nanoparticles. To address these challenges and gaps in our understanding, in this dissertation quantitative approaches and systematic analyses were utilized to 1) evaluate the interactions between phagocytic cells and various PEG coatings on polymeric nanoparticles, 2) determine the prevalence and concentrations of different anti-PEG antibody isotypes amongst the general human population, and 3) apply an alternative approach for PEGylated nanoparticle delivery to tumors. The results indicated that extremely dense PEG coatings (RF/D >> 2.8) are required to effectively minimize nonspecific clearance by immune cells. Using competitive ELISAs and engineered antibody standards, the anti-PEG IgG1-4 and IgM levels in a large number of healthy human samples were quantified, with the majority of samples possessing detectable anti-PEG IgG and/or IgM. Finally, a multistep targeting (i.e., pretargeting) approach was tested for the delivery of biotin PEG-modified nanoparticles to disparate tumor cells in vitro and in vivo. The analytical methodologies and overall findings described here can inform future studies of PEGylated nanoparticle-immune system interactions and nanoparticle targeting strategies.