Maturation of the large ribosomal subunit (LSU) in eukaryotes is a complex and highly coordinated process that requires the concerted action of a large, dynamic, ribonucleoprotein complex, the LSU processome. While we know that >80 ribosome biogenesis factors are required throughout the course of LSU assembly, little is known about how these factors interact with each other within the LSU processome. To interrogate its organization and architecture, we took a systems biology approach and performed a semi-high-throughput, array-based, directed yeast two-hybrid assay. Assaying 4800 protein-protein interactions, we identified 232 high-confidence, binary-interacting protein pairs, representing a fourfold increase from current knowledge. The resulting LSU processome interactome map has enhanced our understanding of the organization and function of the biogenesis factors within the LSU processome, revealing both novel and previously identified subcomplexes and hub proteins, including Nop4.[Keywords: LSU processome; RNA; ribosome; yeast two-hybrid] Supplemental material is available for this article. Eukaryotic ribosomes are complex cellular machines that are comprised of four different ribosomal RNAs (rRNAs) and >70 ribosomal proteins (r-proteins) (Woolford and Baserga 2013). Ribosome assembly begins in the nucleolus with the transcription of the polycistronic pre-rRNA, termed the 35S in Saccharomyces cerevisiae, by RNA polymerase I. The 35S pre-rRNA undergoes numerous cleavage and modification steps to generate the mature 18S, 5.8S, and 25S rRNAs that are assembled with the r-proteins to form the small and large ribosomal subunits. Accurate and efficient production of ribosomes requires the coordinated activity of >150 biogenesis factors, including a number of proteins with enzymatic activity (Fatica and Tollervey 2002; Fromont-Racine et al.