Utilizing a Drosophila model of inclusion body myopathy 3 (IBM‐3), we identified a subset of stress‐response proteins essential for skeletal muscle proteostasis in both health and disease. Inclusion body myopathy 3 (IBM‐3) is a rare, dominant skeletal muscle disease caused by an E706K substitution in the SH1 helix of myosin heavy chain IIa. We produced a model of the human disease in the indirect flight muscle (IFM) of Drosophila. As in the human disease, progressive degradation occurs in muscle expressing the IBM‐3 mutant myosin. Homozygotes assemble defective sarcomeres that accumulate thermally unstable myosin and autophagic vesicles. To further investigate the mechanism of pathogenesis in IBM‐3 muscle fibers, we extracted insoluble proteins from IFM of young and old homozygotes and identified aggregated proteins within inclusions. Quantitative iTRAQ proteomics defined 18 proteins with >1.5‐fold difference in relative abundance at both ages in mutants vs. controls. Two of 6 proteins with increased levels in IBM‐3 have small heat shock protein domains (Hsp20, CG7409), while the 12 proteins with decreased levels include Hsp23, Hsp60, and Trim32 (abba/thin), which has ubiquitin‐protein transferase activity. Using the GAL4‐UAS system for over‐expression or knock down (RNAi), we tested the role of these proteins in wild‐type muscle and determined how altering expression affects the IBM‐3 heterozygote phenotype. While knockdown of Hsp20, Hsp23 or CG7409 yielded normal flight in wild type, Hsp23 or CG7409 over‐expression resulted in age‐dependent reduction in flight ability. Knockdown or over‐expression of these chaperones typically exacerbated the IBM‐3/+ phenotype. As expected, Trim32 knockdown eliminated flight in wild‐type or IBM‐3/+ flies and correlated with less organized, thinner myofibrils. Overexpression of Trim32 also eliminated flight and yielded deteriorating myofibrils that hypercontracted by 3 weeks of age. Remarkably, electron microscopy shows the ultrastructure of this phenotype in IBM‐3/+ resembles the dramatic destruction of myofibrils observed in IBM‐3 homozygotes. Overall, each of the stress response proteins assessed is critical for proper muscle development, performance, or maintenance. Altering levels of these key proteins in muscle fibers challenged with the IBM‐3/+ phenotype has detrimental effects. Our findings add to evidence that expression levels of stress response proteins modulate alternative pathways to respond to challenges to proteostasis.Support or Funding InformationThis work was supported by grants from the Muscular Dystrophy Association (MDA217900)and the National Institutes of Health (NIGMS R37 32443) to S.I.B. The authors acknowledgethe Bloomington Drosophila Stock Center (NIH P40OD018537), the Vienna DrosophilaResource Center and Dr. Richard Cripps (SDSU) for Drosophila stocks.