Regulation of gene expression is fundamental to cell function. Post-transcriptional regulatory mechanisms, those acting directly on messenger RNA (mRNA), are increasingly appreciated for their role in shaping the transcriptome. Post-transcriptional regulation is primarily mediated by the sequence immediately downstream of the coding region, termed the 3′ untranslated region (3′UTR). While it is accepted that 3′UTRs harbor important sequences influencing mRNA decay and translation, the global scope of their regulation is not well understood. Approaches that attempt to ascribe regulatory activity to the 3′UTR by measuring endogenous transcripts suffer from confounding factors such as effects from the 5′ untranslated region (5′UTR) and coding region. Massively parallel reporter assays (MPRAs) mitigate this problem by keeping the 5′UTR and coding region constant; however, technical limitations have restricted this approach to the interrogation of short 3′UTR fragments. This limitation is critical, for cis-regulatory elements within 3′UTRs depend heavily on surrounding sequence context. To address these limitations, I designed and implemented a robust approach to assay full-length human 3′UTRs to quantify their impact on all major modes of quantitative post-transcriptional regulation. To generate reproducible measurements and decrease noise, I integrated internally replicated, single-copy 3′UTR reporters at a constant, stable locus in the genome using site-directed recombination. Using targeted RNA sequencing, polysome profiling, and flow cytometry, I leveraged the power of this system to assay >1,400 human 3′UTR reporters in parallel for their impact on mRNA decay, translational regulation, and total protein output. While 80% of 3′UTRs were expressed over a 5-fold range, the remaining 20% exhibited large differences in expression, with up to a 40-fold difference in expression. I show that this assay can robustly disentangle cumulative regulation (represented by total protein output) into mRNA decay and translation components. I show that mRNA decay and translation are highly correlated, and therefore generally have concordant influence on total protein output on the majority of assayed 3′UTRs. Nevertheless, I also identify 3′UTRs that have high translation rates but do not have pronounced transcript stabilization; such 3′UTRs result in substantially higher protein output than is predicted by their RNA levels. These 3′UTR represent interesting candidates for future studies to better understand 3′UTR-enhanced translation. I demonstrate that 3′UTRs are dynamic in different cellular contexts, and I define relationships between all modes of regulation and various 3′UTR features, including GC content, 3′UTR length, evolutionary conservation, and presence of cis-regulatory elements. Overall, my work represents the most comprehensive investigation of full-length human 3′UTRs and their impact on gene regulation.