Toxic and pathologic protein aggregation is a common feature of neurodegenerative diseases from Alzheimer’s and Parkinson’s diseases to amyotrophic lateral sclerosis (ALS). Chaperone proteins such as the metazoan heat shock protein 70 (Hsp70), Hsp40, and Hsp110 complex can identify these misfolded aggregates and restore them to their functional native states. However, previous studies have used a wide range of chaperone complex components and proportions. Using aggregated firefly luciferase as a model substrate, we set out to optimize the stoichiometric ratio for the disaggregation of these aggregates in vitro. We tested ratios of Hsp70:40:110 from 1:10:1 to 1000:100:1 and used the luminescence of firefly luciferin as a measure of disaggregation activity. With this data, we proceeded to refine the canonical Hsp70/40/110 disaggregase network against fused in sarcoma (FUS), a protein that forms pathologic protein aggregates in ALS and frontotemporal dementia (FTD). We used absorbance measurements at 395 nm from a high‐throughput plate reader to determine the turbidity levels of FUS aggregates with various combinations of Hsp70/40/110. Together, our findings support the existence of an ideal Hsp70/40/110 system which provides a framework to optimize the disassembly of protein aggregates in other neurodegenerative diseases.