A hallmark of amyotrophic lateral sclerosis (ALS) pathology is the accumulation of ubiquitylated protein inclusions within motor neurons. Recent studies suggest the sequestration of ubiquitin (Ub) into inclusions reduces the availability of free Ub, which is essential for cellular function and survival. However, the dynamics of the Ub landscape in ALS have not yet been described. Here, we show that Ub homeostasis is altered in a cell model of ALS induced by expressing mutant SOD1 (SOD1A4V). By monitoring the distribution of Ub in cells expressing SOD1A4V, we show that Ub is present at the earliest stages of SOD1A4V aggregation, and that cells containing SOD1A4V aggregates have greater ubiquitin-proteasome system (UPS) dysfunction. Furthermore, SOD1A4V aggregation is associated with the redistribution of Ub and depletion of the free Ub pool. Ubiquitomics analysis indicates that expression of SOD1A4V is associated with a shift of Ub to a pool of supersaturated proteins, including those associated with oxidative phosphorylation and metabolism, corresponding with altered mitochondrial morphology and function. Taken together, these results suggest that misfolded SOD1 contributes to UPS dysfunction and that Ub homeostasis is an important target for monitoring pathological changes in ALS.