Protein misfolding underpins numerous fatal neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Parkinson's disease (PD). Hsp104, a conserved hexameric AAA+ protein-remodeling factor from yeast, solubilizes disordered aggregates and amyloid. Hsp104 can disaggregate misfolded proteins implicated in neurodegeneration, however this activity is weak, which has prompted efforts to engineer Hsp104 variants with enhanced activity. Many potentiated Hsp104 variants have been generated, however application of these variants in mammalian systems has stalled due to suboptimal substrate specificity and off-target effects of the variants. Here, we present a high-throughput approach to identify substrate-optimized enhanced Hsp104 variants that solubilize preformed α-syn and TDP-43 aggregates with diminished off-target toxicity in mammalian cells. Mechanistic studies of these new variants also reveal new insights into Hsp104 function and potentiation. We anticipate that our screening approach could be broadly applied to a range of protein engineering targets and establish that the new Hsp104 variants we have uncovered have key improvements facilitating their application in mammalian cells.