Nearly half of human genes arose from ancestral versions dating back to the Last Eukaryotic Common Ancestor (LECA) roughly 1.5 billion years ago. Many current genetic diseases and traits stem from these ancient molecular systems, yet how these protein networks guide human development and physiology remains poorly understood.  Most proteins function as components of multi-protein complexes, and because such proteins co-evolve, studies of protein complexes in a wide array of organisms can provide insights into these systems. I’ll describe our attempts to define and discover deeply conserved eukaryotic multi-protein complexes using high-throughput protein mass spectrometry to measure protein assemblies shared across the eukaryotic tree of life, including thousands of shotgun proteomics experiments across humans and a variety of animals, plants, and representative unicellular ciliates and diatoms. The resulting interactome describes the biochemical organization of LECA, revealing both known and new assemblies. I’ll discuss how, by exploring these ancient protein interactions, we’ve found new human gene/disease relationships for bone density and congenital birth defects, demonstrating the value of ancestral protein interactions for guiding functional genetics today.