Mitochondrial dysfunction and quality control has become a central theme in neurodegenerative diseases. Ubiquitination of mitochondrial membrane proteins by E3 ubiquitin ligase Parkin or other ubiquitin ligases is a critical step preceding mitophagy, a selective type of autophagy that guards mitochondrial quality control. We recently discovered that mitophagy can be enhanced by reducing either the levels or activity of the deubiquitinating enzyme USP14. Knocking down USP14 in two established fly models of mitochondrial dysfunction, restored mitochondria function and ultrastructure, and brain dopamine levels. Remarkably, at the systemic level it extended the flies’ lifespan and rescued climbing behavior. An orally bioavailable small-molecule inhibitor of USP14, IU1, had similarly beneficial effects on these mutant flies. Studies on the effects of USP14 inhibition in mammalian neurons have not yet been conducted. To close this gap, we exploited iNeurons differentiated from human embryonic stem cells (hESCs) to investigate the potential beneficial effect of enhancing mitophagy in models known to accumulate dysfunctional mitochondria. We found that USP14 inhibition is beneficial in these neurons of human origin. We also investigated the underlying mechanism of suppression, using acute, time-resolved models of USP14 inhibition in iNeurons in conjunction with state-of-the-art quantitative global proteomics, with a key goal being to identify the substrate or substrates of USP14 whose deubiquitination negatively regulates basal mitophagy. Our study provides new mechanistic insights into mitochondrial maintenance, which is relevant to several major neurodegenerative diseases associated with mitochondrial dysfunction.