USP7 is a deubiquitylating enzyme, a hub of a multi-nodal network, that is involved in tumor suppression, neuro)development, chromatin regulation and the DNA damage response. How USP7 regulates these diverse pathways is still unclear. Here, we used data-independent acquisition and label free quantitation mass spectrometry (DIA-LFQ-MS) to profile the proteome-wide impact of USP7 on substrate de-ubiquitylation and overall protein abundance. We identified proteins associated with endogenous USP7 by immunopurification followed by DIA-LFQ-MS. Integration of our new results with earlier interactomes of epitope-tagged USP7 yielded a consensus set of high-confidence partner proteins and substrates. Using specific enrichment of tryptic K-GG peptides, we mapped proteome-wide changes in ubiquitinome dynamics following inhibition of USP7. Combining unbiased proteome-wide and targeted quantitative mass spectrometry revealed that deubiquitylation by USP7 can have very different effects on the stability of distinct substrates and suggests that USP7’s activity profile is thus substrate-dependent rather than an intrinsic enzymatic property. Thus, in addition to providing a proteome-wide map of USP7 target sites, our multi-angle proteomics approach shows that the effects of USP7-mediated deubiquitination on its targets are remarkably variable and substrate-specific. Finally, we show based on these obtained detailed molecular insights how USP7 connects various neurodevelopmental syndromes in addition to Hao Fountain syndrome.