2020 The Author(s) A major feature of amyotrophic lateral sclerosis (ALS) pathology is the accumulation of ubiquitin (Ub) into intracellular inclusions. This sequestration of Ub may reduce the availability of free Ub, disrupting Ub homeostasis and ultimately compromising cellular function and survival. We previously reported significant disturbance of Ub homeostasis in neuronal-like cells expressing mutant SOD1. Here, we show that Ub homeostasis is also perturbed in neuronal-like cells expressing either TDP-43 or FUS. The expression of mutant TDP-43 and mutant FUS led to UPS dysfunction, which was associated with a redistribution of Ub and depletion of the free Ub pool. Redistribution of Ub is also a feature of sporadic ALS, with an increase in Ub signal associated with inclusions and no compensatory increase in Ub expression. Together, these findings suggest that alterations to Ub homeostasis caused by the misfolding and aggregation of ALS-associated proteins play an important role in the pathogenesis of ALS.