Mind bomb 1 (Mib1) is an E3 ubiquitin-ligase that is essential for overall metazoan development, including multiple stages of neuronal development. It is located in puncta throughout neurons, including near post synaptic densities (PSD), and has been shown to participate in several signaling pathways via its E3 ligase catalytic activity. The most well-characterized of these is the Notch signaling pathway, in which Mib1 facilitates Delta/Serrate/LAG-2 (DSL) ligand endocytosis and activation, allowing cell-cell communication to determine neuronal versus glial cell fate. This, however, is not the limit of Mib1 activity in the developing nervous system, as it also contributes to cell polarity, neurite outgrowth, and long-term potentiation (LTP), but it has not been demonstrated to affect dendritic spine development. We therefore sought to comprehensively characterize the Mib1 interactome and study its potential function in dendritic spine morphogenesis. We utilized a novel sequential elution method from Mib1 affinity purification to recover Mib1 binding proteins with both deep coverage and the ability to distinguish between high affinity binding partners from low affinity binding partners. This procedure revealed 837 potential binding partners, distinguished 72 from these as very-high confidence, and a further 387 as high confidence of interaction. Included in these were many proteins previously demonstrated to interact with Mib1, as well as 5 proteins of particular interest to us: Usp9x, a deubiquitinase; alpha-, beta-, and delta-catenins; regulators of Wnt signaling; and CDKL5, which is mutated in EIEE2, a severe form of mental retardation. We demonstrated that Mib1 downregulates CDKL5, limits its effects on dendritic spine outgrowth, and inhibits spine outgrowth itself. These data further elaborate upon the signaling networks and biological functions influenced by this critical protein and expand our understanding of the signaling networks involved in neuronal development.