Human 14-3-3 proteins are a family of seven highly conserved phospho-serine/threonine binding domains. They interact with hundreds/thousands of structurally and functionally diverse protein clients, thereby acting as central hubs of many signaling networks. However, how each 14-3-3 paralog differs in client specificity is not known for most clients because interaction specificity differences have never been quantitatively assessed on a large scale for the full complement of 14-3-3 paralogs. Furthermore, due to the lack of systematic functional screens, it is not known how most client proteins are regulated by 14-3-3s. Here, I used affinity purification coupled to mass spectrometry (AP-MS), proximity biotinylation (BioID2), and LUminescence-based Mammalian IntERactome profiling (LUMIER) to generate quantitative and directly comparable interactomes of all seven human 14-3-3 paralogs. I also used a high-content immunofluorescent imaging approach to uncover the effect of disrupting 14-3-3 interactions on client protein localization. The resulting networks revealed that all paralogs, with the exception of 14-3-3σ, have extremely similar or nearly identical interaction specificities. Furthermore, I found that loss of 14-3-3 binding leads to coalescence of a large fraction of clients into discrete foci or clusters in a client-specific manner, suggesting a central chaperone-like function for 14-3-3 proteins. Consistent with this, engraftment of 14-3-3 binding motifs to non-clients suppressed their aggregation or phase separation. This is also supported by the enrichment of features in 14-3-3 interactors that promote aggregation and phase separation. Finally, I showed that 14-3-3 proteins negatively regulate the localization of the RNA-binding protein SAMD4A to cytoplasmic granules and suppress its activity as a translational repressor. Ultimately, I was able to generate the most comprehensive framework for deciphering differences in interaction preferences between 14-3-3 paralogs. Furthermore, my work was able to greatly expand on the functional consequences of 14-3-3 binding in human cells, revealing that 14-3-3 proteins have a more prominent role as chaperone-like molecules than previously thought.